llc pk1  (ATCC)


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    Structured Review

    ATCC llc pk1
    Role of p38 in inhibiting PDCoV replication and alleviating PDCoV-induced apoptosis by EP in <t>LLC-PK1</t> cells. A The efficiency of p38 siRNA was evaluated by Western blot. LLC-PK1 cells were transfected with the indicated siRNA. At 24 h post-transfection, the expression of p38 was analyzed by Western blot. Results were presented as the ratio of p38 band intensity to β-actin band intensity. B LLC-PK1 cells grown in 6-well plates were transfected with p38 siRNA #1 or NC siRNA for 24 h, and then infected with 2 MOI PDCoV or mock-infected in the absence or presence EP (150 μM). After PDCoV adsorption for 1 h, the cells were further cultured in fresh medium in the absence or presence EP (150 μM). Western blot analysis of proteins from indicated LLC-PK1 cells probed with the anti-p-p38, anti-p38, anti-cleaved caspase-3, anti-caspase-3, anti-Bax, anti-Bcl-2 and anti-PDCoV N antibody. C – I Results are presented as the ratio of target protein band intensity to β-actin band intensity. Data are presented as the mean ± SEM. * P
    Llc Pk1, supplied by ATCC, used in various techniques. Bioz Stars score: 95/100, based on 38 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "Antiviral effects of ergosterol peroxide in a pig model of porcine deltacoronavirus (PDCoV) infection involves modulation of apoptosis and tight junction in the small intestine"

    Article Title: Antiviral effects of ergosterol peroxide in a pig model of porcine deltacoronavirus (PDCoV) infection involves modulation of apoptosis and tight junction in the small intestine

    Journal: Veterinary Research

    doi: 10.1186/s13567-021-00955-5

    Role of p38 in inhibiting PDCoV replication and alleviating PDCoV-induced apoptosis by EP in LLC-PK1 cells. A The efficiency of p38 siRNA was evaluated by Western blot. LLC-PK1 cells were transfected with the indicated siRNA. At 24 h post-transfection, the expression of p38 was analyzed by Western blot. Results were presented as the ratio of p38 band intensity to β-actin band intensity. B LLC-PK1 cells grown in 6-well plates were transfected with p38 siRNA #1 or NC siRNA for 24 h, and then infected with 2 MOI PDCoV or mock-infected in the absence or presence EP (150 μM). After PDCoV adsorption for 1 h, the cells were further cultured in fresh medium in the absence or presence EP (150 μM). Western blot analysis of proteins from indicated LLC-PK1 cells probed with the anti-p-p38, anti-p38, anti-cleaved caspase-3, anti-caspase-3, anti-Bax, anti-Bcl-2 and anti-PDCoV N antibody. C – I Results are presented as the ratio of target protein band intensity to β-actin band intensity. Data are presented as the mean ± SEM. * P
    Figure Legend Snippet: Role of p38 in inhibiting PDCoV replication and alleviating PDCoV-induced apoptosis by EP in LLC-PK1 cells. A The efficiency of p38 siRNA was evaluated by Western blot. LLC-PK1 cells were transfected with the indicated siRNA. At 24 h post-transfection, the expression of p38 was analyzed by Western blot. Results were presented as the ratio of p38 band intensity to β-actin band intensity. B LLC-PK1 cells grown in 6-well plates were transfected with p38 siRNA #1 or NC siRNA for 24 h, and then infected with 2 MOI PDCoV or mock-infected in the absence or presence EP (150 μM). After PDCoV adsorption for 1 h, the cells were further cultured in fresh medium in the absence or presence EP (150 μM). Western blot analysis of proteins from indicated LLC-PK1 cells probed with the anti-p-p38, anti-p38, anti-cleaved caspase-3, anti-caspase-3, anti-Bax, anti-Bcl-2 and anti-PDCoV N antibody. C – I Results are presented as the ratio of target protein band intensity to β-actin band intensity. Data are presented as the mean ± SEM. * P

    Techniques Used: Western Blot, Transfection, Expressing, Infection, Adsorption, Cell Culture

    2) Product Images from "Porcine deltacoronavirus nsp15 antagonizes interferon-β production independently of its endoribonuclease activity"

    Article Title: Porcine deltacoronavirus nsp15 antagonizes interferon-β production independently of its endoribonuclease activity

    Journal: Molecular Immunology

    doi: 10.1016/j.molimm.2019.07.003

    PDCoV nsp15 antagonizes SEV-induced IFN-β production. ( A , C ) LLC-PK1 cells (A) or HEK-293T cells (C) cultured in 24-well plates were co-transfected with IFN-β-Luc and pRL-TK together with increasing amounts (0.1, 0.5, and 1.0 μg) of pCAGGS-HA-nsp15 or empty vector. After 24 h, the cells were mock-infected or infected with SEV (10 hemagglutinating activity units/well) for 12 h and subjected to a dual-luciferase reporter assay. The relative firefly luciferase activity was normalized to the Renilla luciferase activity with the untreated empty vector control value set to 1. ( B , D ) LLC-PK1 cells (B) or HEK-293T cells (D) cultured in 24-well plates were co-transfected with 1.0 μg of pCAGGS-HA-nsp15 or empty vector for 24 h, and then left untreated or infected with SEV (10 hemagglutinating activity units/well). At 8 h after infection, the cells were collected, and total RNA was extracted to detect the expression levels of IFN-β and GAPDH by SYBR Green PCR assay. ( E , F ) LLC-PK1 (E) or HEK-293T (F) cells were transfected with 1.0 μg of pCAGGS-HA-nsp15 or empty vector. At 24 h after transfection, both cell lines were infected with SEV for 12 h and the cell supernatants were collected. The UV-irradiated cell supernatants were overlaid onto fresh LLC-PK1 or HEK-293T cells in 24-well plates. After 24 h of incubation, cells were infected with VSV-GFP for 12 h. The replication of VSV-GFP was detected via fluorescence microscopy. Data are representative of three independent experiments. ***, p
    Figure Legend Snippet: PDCoV nsp15 antagonizes SEV-induced IFN-β production. ( A , C ) LLC-PK1 cells (A) or HEK-293T cells (C) cultured in 24-well plates were co-transfected with IFN-β-Luc and pRL-TK together with increasing amounts (0.1, 0.5, and 1.0 μg) of pCAGGS-HA-nsp15 or empty vector. After 24 h, the cells were mock-infected or infected with SEV (10 hemagglutinating activity units/well) for 12 h and subjected to a dual-luciferase reporter assay. The relative firefly luciferase activity was normalized to the Renilla luciferase activity with the untreated empty vector control value set to 1. ( B , D ) LLC-PK1 cells (B) or HEK-293T cells (D) cultured in 24-well plates were co-transfected with 1.0 μg of pCAGGS-HA-nsp15 or empty vector for 24 h, and then left untreated or infected with SEV (10 hemagglutinating activity units/well). At 8 h after infection, the cells were collected, and total RNA was extracted to detect the expression levels of IFN-β and GAPDH by SYBR Green PCR assay. ( E , F ) LLC-PK1 (E) or HEK-293T (F) cells were transfected with 1.0 μg of pCAGGS-HA-nsp15 or empty vector. At 24 h after transfection, both cell lines were infected with SEV for 12 h and the cell supernatants were collected. The UV-irradiated cell supernatants were overlaid onto fresh LLC-PK1 or HEK-293T cells in 24-well plates. After 24 h of incubation, cells were infected with VSV-GFP for 12 h. The replication of VSV-GFP was detected via fluorescence microscopy. Data are representative of three independent experiments. ***, p

    Techniques Used: Cell Culture, Transfection, Plasmid Preparation, Infection, Activity Assay, Luciferase, Reporter Assay, Expressing, SYBR Green Assay, Polymerase Chain Reaction, Irradiation, Incubation, Fluorescence, Microscopy

    PDCoV nsp15 impairs SEV-induced activation of NF-κB. ( A – D ) LLC-PK1 cells (A, C) or HEK-293T cells (B, D) grown in 24-well plates were co-transfected with NF-κB-Luc (A, B) or IRF3-Luc (C, D) and pRL-TK together with increasing quantities (0.1, 0.5, and 1.0 μg) of PDCoV nsp15 expression plasmid for 24 h, followed by infection with SEV or mock-infection for 12 h before luciferase reporter assays were performed. The averages of data from three independent experiments are shown. ***, p
    Figure Legend Snippet: PDCoV nsp15 impairs SEV-induced activation of NF-κB. ( A – D ) LLC-PK1 cells (A, C) or HEK-293T cells (B, D) grown in 24-well plates were co-transfected with NF-κB-Luc (A, B) or IRF3-Luc (C, D) and pRL-TK together with increasing quantities (0.1, 0.5, and 1.0 μg) of PDCoV nsp15 expression plasmid for 24 h, followed by infection with SEV or mock-infection for 12 h before luciferase reporter assays were performed. The averages of data from three independent experiments are shown. ***, p

    Techniques Used: Activation Assay, Transfection, Expressing, Plasmid Preparation, Infection, Luciferase

    PDCoV nsp15 inhibits IFN-β production independently of its endoribonuclease activity. LLC-PK1 cells ( A ) or HEK-293T cells ( B ) cultured in 24-well plates were transfected with 1.0 μg of expression plasmid (PDCoV nsp15, H219A, H234A, or K269A) or empty vector, along with IFN-β-Luc and pRL-TK for 24 h. The cells were then mock-infected or infected with SEV for 12 h and subjected to dual-luciferase reporter assays. Data are means ± SD from three independent experiments. ***, p
    Figure Legend Snippet: PDCoV nsp15 inhibits IFN-β production independently of its endoribonuclease activity. LLC-PK1 cells ( A ) or HEK-293T cells ( B ) cultured in 24-well plates were transfected with 1.0 μg of expression plasmid (PDCoV nsp15, H219A, H234A, or K269A) or empty vector, along with IFN-β-Luc and pRL-TK for 24 h. The cells were then mock-infected or infected with SEV for 12 h and subjected to dual-luciferase reporter assays. Data are means ± SD from three independent experiments. ***, p

    Techniques Used: Activity Assay, Cell Culture, Transfection, Expressing, Plasmid Preparation, Infection, Luciferase

    PDCoV nsp15 mutants lacking endoribonuclease activity also significantly inhibit SEV-induced activation of NF-κB. ( A , B ) LLC-PK1 cells (A) or HEK-293T cells (B) grown in 24-well plates were co-transfected with NF-κB–Luc together with pRL-TK and 1.0 μg of expression plasmid (PDCoV nsp15, H219A, H234A, K269A) or empty vector for 24 h, followed by stimulation with SEV for 12 h before luciferase reporter assays were performed. ***, p
    Figure Legend Snippet: PDCoV nsp15 mutants lacking endoribonuclease activity also significantly inhibit SEV-induced activation of NF-κB. ( A , B ) LLC-PK1 cells (A) or HEK-293T cells (B) grown in 24-well plates were co-transfected with NF-κB–Luc together with pRL-TK and 1.0 μg of expression plasmid (PDCoV nsp15, H219A, H234A, K269A) or empty vector for 24 h, followed by stimulation with SEV for 12 h before luciferase reporter assays were performed. ***, p

    Techniques Used: Activity Assay, Activation Assay, Transfection, Expressing, Plasmid Preparation, Luciferase

    3) Product Images from "Development and utilization of an infectious clone for porcine deltacoronavirus strain USA/IL/2014/026"

    Article Title: Development and utilization of an infectious clone for porcine deltacoronavirus strain USA/IL/2014/026

    Journal: Virology

    doi: 10.1016/j.virol.2020.11.002

    Generation and characterization of PDCoV mutant viruses. (A) Two mutant PDCoVs were generated in this study. icEnUmut: Histidine-219 of the endoribonuclease (EndoU) of PDCoV was mutated to Alanine (H219A). icDelNS6/nG: the coding sequence of ns6 was replaced with neonGreen (nGreen) sequence. The TRS of ns6 and N gene are shown and boxed. A 20 nt coding sequence of ns6 at the 3′ end was retained to maintain the potential secondary structure of the TRS of N gene. (B) Representative plaques of the parental strain-, icPDCoV-, icEnUmut-, and icDelNS6/nG-infected PK1 cells at 32 h post-infection. (C) Growth kinetics of icPDCoV and mutant viruses. PK1 cells were infected at a dose of 0.1 TCID 50 per cell. Cell culture supernatant was collected at the indicated time points and subject to a TCID 50 assay in PK1 cells. Error bars represent mean ± standard deviation. (D) icDelNS6/nG infection in PK1 cells caused CPE (Bright) and yielded green signal (nGreen) under a fluorescence microscope. (E) Western blotting detection of the expression of N protein using mouse anti -N monoclonal antibody (mAb). β-actin served as a loading control. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
    Figure Legend Snippet: Generation and characterization of PDCoV mutant viruses. (A) Two mutant PDCoVs were generated in this study. icEnUmut: Histidine-219 of the endoribonuclease (EndoU) of PDCoV was mutated to Alanine (H219A). icDelNS6/nG: the coding sequence of ns6 was replaced with neonGreen (nGreen) sequence. The TRS of ns6 and N gene are shown and boxed. A 20 nt coding sequence of ns6 at the 3′ end was retained to maintain the potential secondary structure of the TRS of N gene. (B) Representative plaques of the parental strain-, icPDCoV-, icEnUmut-, and icDelNS6/nG-infected PK1 cells at 32 h post-infection. (C) Growth kinetics of icPDCoV and mutant viruses. PK1 cells were infected at a dose of 0.1 TCID 50 per cell. Cell culture supernatant was collected at the indicated time points and subject to a TCID 50 assay in PK1 cells. Error bars represent mean ± standard deviation. (D) icDelNS6/nG infection in PK1 cells caused CPE (Bright) and yielded green signal (nGreen) under a fluorescence microscope. (E) Western blotting detection of the expression of N protein using mouse anti -N monoclonal antibody (mAb). β-actin served as a loading control. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

    Techniques Used: Mutagenesis, Generated, Sequencing, Infection, Cell Culture, Standard Deviation, Fluorescence, Microscopy, Western Blot, Expressing

    Characterization of host immune responses in icPDCoV- and mutant viruses-infected PK1 cells. Cells were infected at a dose of 0.1 TCID 50 per cell and were harvested for total RNA extraction at indicated time points. The relative mRNA level of (A) IFN-β, N gene, (B) ISGs (OAS1, ISG15, ISG54), and TNF-α were measured using RT-qPCR. The experiment was performed three times, and the representative data are shown. Error bars represent mean ± SD and the relative fold-changes were shown above the error bars.
    Figure Legend Snippet: Characterization of host immune responses in icPDCoV- and mutant viruses-infected PK1 cells. Cells were infected at a dose of 0.1 TCID 50 per cell and were harvested for total RNA extraction at indicated time points. The relative mRNA level of (A) IFN-β, N gene, (B) ISGs (OAS1, ISG15, ISG54), and TNF-α were measured using RT-qPCR. The experiment was performed three times, and the representative data are shown. Error bars represent mean ± SD and the relative fold-changes were shown above the error bars.

    Techniques Used: Mutagenesis, Infection, RNA Extraction, Quantitative RT-PCR

    Adaptive mutations allow rescue of infectious PDCoV that produces observable cytopathic effect in PK1 cells. In order to obtain cell culture adaptive mutation(s), the parental strain (P1) was passaged for 30 times (P30) in PK1 cells. (A) Growth kinetics analysis of P1 and P30 indicates P30 grew more rapidly and reached peak titer earlier than P1. (B) Full-genome sequencing revealed six nonsynonymous nucleotide substitutions in the genome of the P30 virus. (C) Four of the six substitutions reside in the spike sequence and are indicated in the protein structure of PDCoV spike (PDB#: 6b7n). (D) Incorporating all four (icP30) or one (H236Y) of the spike substitutions (icPDCoV) allows the recovered infectious clone virus to produce CPE in PK1 cells.
    Figure Legend Snippet: Adaptive mutations allow rescue of infectious PDCoV that produces observable cytopathic effect in PK1 cells. In order to obtain cell culture adaptive mutation(s), the parental strain (P1) was passaged for 30 times (P30) in PK1 cells. (A) Growth kinetics analysis of P1 and P30 indicates P30 grew more rapidly and reached peak titer earlier than P1. (B) Full-genome sequencing revealed six nonsynonymous nucleotide substitutions in the genome of the P30 virus. (C) Four of the six substitutions reside in the spike sequence and are indicated in the protein structure of PDCoV spike (PDB#: 6b7n). (D) Incorporating all four (icP30) or one (H236Y) of the spike substitutions (icPDCoV) allows the recovered infectious clone virus to produce CPE in PK1 cells.

    Techniques Used: Cell Culture, Mutagenesis, Sequencing

    Characterization of the rescued PDCoV from the infectious clone. (A) Representative plaques of mock-, the parental strain-, and icPDCoV-infected LLC-PK1 cells at 40 h post-infection. (B) PCR fragment amplified from the parental virus was digested by the Sap I enzyme, while the PCR product derived from icPDCoV was resistant to Sap I-digestion. (C) Growth kinetics of the parental strain (Parental) and infectious clone strain (icPDCoV) in PK1 cells. (D) Detection of N protein expression in parental- or icPDCoV-infected PK1 cells using an immunofluorescence assay with a mouse anti -N monoclonal antibody.
    Figure Legend Snippet: Characterization of the rescued PDCoV from the infectious clone. (A) Representative plaques of mock-, the parental strain-, and icPDCoV-infected LLC-PK1 cells at 40 h post-infection. (B) PCR fragment amplified from the parental virus was digested by the Sap I enzyme, while the PCR product derived from icPDCoV was resistant to Sap I-digestion. (C) Growth kinetics of the parental strain (Parental) and infectious clone strain (icPDCoV) in PK1 cells. (D) Detection of N protein expression in parental- or icPDCoV-infected PK1 cells using an immunofluorescence assay with a mouse anti -N monoclonal antibody.

    Techniques Used: Infection, Polymerase Chain Reaction, Amplification, Derivative Assay, Expressing, Immunofluorescence

    4) Product Images from "A multi-omics study to investigate tacrolimus nephrotoxicity mechanisms"

    Article Title: A multi-omics study to investigate tacrolimus nephrotoxicity mechanisms

    Journal: bioRxiv

    doi: 10.1101/2021.07.29.454229

    LLC-PK1 viability after incubation with 0.5% ethanol (control (C)) or 5µM of tacrolimus (TAC) for 24h, 48h and 72h, assessed using the MTS viability assay (n=18, left graph) and annexin 5/ 7AAD staining (n=5, right graph). Graphs represent the % of cell viability with tacrolimus as compared to control for each incubation duration. Statistical analysis with the t-test. *** p
    Figure Legend Snippet: LLC-PK1 viability after incubation with 0.5% ethanol (control (C)) or 5µM of tacrolimus (TAC) for 24h, 48h and 72h, assessed using the MTS viability assay (n=18, left graph) and annexin 5/ 7AAD staining (n=5, right graph). Graphs represent the % of cell viability with tacrolimus as compared to control for each incubation duration. Statistical analysis with the t-test. *** p

    Techniques Used: Incubation, Viability Assay, Staining

    5) Product Images from "Environmental Xenobiotics and the Antihormones Cyproterone Acetate and Spironolactone Use the Nuclear Hormone Pregnenolone X Receptor to Activate the CYP3A23 Hormone Response Element"

    Article Title: Environmental Xenobiotics and the Antihormones Cyproterone Acetate and Spironolactone Use the Nuclear Hormone Pregnenolone X Receptor to Activate the CYP3A23 Hormone Response Element

    Journal: Molecular pharmacology

    doi:

    Steroids activate the CYP3A23 DR-3 in LLC-PK1 cells co-transfected with PXR. LLC-PK1 cells co-transfected with mouse PXR, (CYP3A23) 2 -tk-CAT, and TK-LUC were treated with 50 μ M of the indicated steroids ( control , no steroid treatment) for 24 hr and cell extracts were assayed for CAT and Luciferase activities. Data was graphed as -fold increase over untreated control and represented CAT activity in treated cells normalized to co-transfected TK-LUC in the same sample; this treatment (CAT/LUC) value was divided by the mean (CAT/LUC) value from 3–4 control wells. The control value was 0.974 (±) 0.087. The values represent means ± standard deviation from a representative experiment (repeated two to four times) with three to four replicates per treatment per experiment.
    Figure Legend Snippet: Steroids activate the CYP3A23 DR-3 in LLC-PK1 cells co-transfected with PXR. LLC-PK1 cells co-transfected with mouse PXR, (CYP3A23) 2 -tk-CAT, and TK-LUC were treated with 50 μ M of the indicated steroids ( control , no steroid treatment) for 24 hr and cell extracts were assayed for CAT and Luciferase activities. Data was graphed as -fold increase over untreated control and represented CAT activity in treated cells normalized to co-transfected TK-LUC in the same sample; this treatment (CAT/LUC) value was divided by the mean (CAT/LUC) value from 3–4 control wells. The control value was 0.974 (±) 0.087. The values represent means ± standard deviation from a representative experiment (repeated two to four times) with three to four replicates per treatment per experiment.

    Techniques Used: Transfection, Luciferase, Activity Assay, Standard Deviation

    6) Product Images from "Functional reconstitution, membrane targeting, genomic structure, and chromosomal localization of a human urate transporter"

    Article Title: Functional reconstitution, membrane targeting, genomic structure, and chromosomal localization of a human urate transporter

    Journal: Journal of Clinical Investigation

    doi:

    Confocal microscopy of stably transfected LLC-PK1 cells expressing EGFP fused to the carboxy terminus of the urate transporter (hUAT-EGFP). ( a ) Sagittal confocal image and ( b ) horizontal confocal image of cells grown for 6 days on Transwell clear permeable filter supports.
    Figure Legend Snippet: Confocal microscopy of stably transfected LLC-PK1 cells expressing EGFP fused to the carboxy terminus of the urate transporter (hUAT-EGFP). ( a ) Sagittal confocal image and ( b ) horizontal confocal image of cells grown for 6 days on Transwell clear permeable filter supports.

    Techniques Used: Confocal Microscopy, Stable Transfection, Transfection, Expressing

    Bright-field, fluorescent, and confocal microscopy of nonpermeabilized and permeabilized stable pools of LLC-PK1 cells expressing hUAT/FLAG chimeric proteins. ( a – d and i ) Cells transfected with constructs containing FLAG on the amino terminus of hUAT. ( e – h and j ) Cells transfected with constructs containing FLAG on the carboxy terminus of hUAT. ( a , c , e , and g ) Bright-field images of nonpermeabilized ( a and e ) and permeabilized ( c and g ) cells. ( b , d , f , and h ) Fluorescent images of nonpermeabilized ( b and f ) and permeabilized ( d and h ) cells. ( i and j ) Confocal images of permeabilized cells.
    Figure Legend Snippet: Bright-field, fluorescent, and confocal microscopy of nonpermeabilized and permeabilized stable pools of LLC-PK1 cells expressing hUAT/FLAG chimeric proteins. ( a – d and i ) Cells transfected with constructs containing FLAG on the amino terminus of hUAT. ( e – h and j ) Cells transfected with constructs containing FLAG on the carboxy terminus of hUAT. ( a , c , e , and g ) Bright-field images of nonpermeabilized ( a and e ) and permeabilized ( c and g ) cells. ( b , d , f , and h ) Fluorescent images of nonpermeabilized ( b and f ) and permeabilized ( d and h ) cells. ( i and j ) Confocal images of permeabilized cells.

    Techniques Used: Confocal Microscopy, Expressing, Transfection, Construct

    Western blot of surface biotinylated proteins harvested from stable pools of LLC-PK1 cells expressing hUAT/EGFP chimeric proteins or EGFP without hUAT. Lanes 1–3 contain eluates from LLC-PK1 cells transfected with constructs containing EGFP on the amino terminus of hUAT (lane 1), the carboxy terminus of hUAT (lane 2), and EGFP alone (lane 3). Lanes 1 and 3 contain 5 μl eluate; lane 2 contains approximately 0.3 μl eluate.
    Figure Legend Snippet: Western blot of surface biotinylated proteins harvested from stable pools of LLC-PK1 cells expressing hUAT/EGFP chimeric proteins or EGFP without hUAT. Lanes 1–3 contain eluates from LLC-PK1 cells transfected with constructs containing EGFP on the amino terminus of hUAT (lane 1), the carboxy terminus of hUAT (lane 2), and EGFP alone (lane 3). Lanes 1 and 3 contain 5 μl eluate; lane 2 contains approximately 0.3 μl eluate.

    Techniques Used: Western Blot, Expressing, Transfection, Construct

    7) Product Images from "Characterization, Pathogenicity and Protective efficacy of a Cell Culture-Derived Porcine Deltacoronavirus"

    Article Title: Characterization, Pathogenicity and Protective efficacy of a Cell Culture-Derived Porcine Deltacoronavirus

    Journal: Virus Research

    doi: 10.1016/j.virusres.2020.197955

    (a) Immunofluorescence detection results for the sixth passage (P10) of the cell culture-adapted PDCoV strain CH/XJYN/2016 in infected LLC-PK cells at 0, 12, 24 and 36 h after inoculation (400 ×). PDCoV antigens and nuclei were detected with mouse anti-PDCoV N protein monoclonal antibodies (McAbs) and 4',6-diamidino-2-phenylindole (DAPI), respectively. LLC-PK cells were fixed and strained with mouse anti-PDCoV N and FITC-goat anti-mouse IgG at 12, 24 and 36 h post-infection (green), nuclei were counterstained with DAPI (blue) ; (b) Electron micrograph of PDCoV virions in cell culture media of infected LLC-PK cells. The arrow indicates the crown-shaped projections on the virus surface; (c) Viral titers of the PDCoV strain CH/XJYN/2016 propagated in LLC-PK cells after serial passage. All the results of a representative experiment performed with triplicate samples are shown.
    Figure Legend Snippet: (a) Immunofluorescence detection results for the sixth passage (P10) of the cell culture-adapted PDCoV strain CH/XJYN/2016 in infected LLC-PK cells at 0, 12, 24 and 36 h after inoculation (400 ×). PDCoV antigens and nuclei were detected with mouse anti-PDCoV N protein monoclonal antibodies (McAbs) and 4',6-diamidino-2-phenylindole (DAPI), respectively. LLC-PK cells were fixed and strained with mouse anti-PDCoV N and FITC-goat anti-mouse IgG at 12, 24 and 36 h post-infection (green), nuclei were counterstained with DAPI (blue) ; (b) Electron micrograph of PDCoV virions in cell culture media of infected LLC-PK cells. The arrow indicates the crown-shaped projections on the virus surface; (c) Viral titers of the PDCoV strain CH/XJYN/2016 propagated in LLC-PK cells after serial passage. All the results of a representative experiment performed with triplicate samples are shown.

    Techniques Used: Immunofluorescence, Cell Culture, Infection

    8) Product Images from "The Porcine Deltacoronavirus Replication Organelle Comprises Double-Membrane Vesicles and Zippered Endoplasmic Reticulum with Double-Membrane Spherules"

    Article Title: The Porcine Deltacoronavirus Replication Organelle Comprises Double-Membrane Vesicles and Zippered Endoplasmic Reticulum with Double-Membrane Spherules

    Journal: Viruses

    doi: 10.3390/v11111030

    Sites of PDCoV RNA synthesis were associated with viral N protein, but not the endoplasmic reticulum (ER). LLC-PK1 cells were infected or mock-infected. Thirty minutes prior to fixation, cells were incubated with 2 mM 5-Bromouridine (BrU) and 15 μM actinomycin D. Cells were fixed and labelled with anti-BrdU (green) and either anti-N or anti-ER antibodies (red). Nuclei were stained with DAPI (blue), scale bar indicates 10 µm.
    Figure Legend Snippet: Sites of PDCoV RNA synthesis were associated with viral N protein, but not the endoplasmic reticulum (ER). LLC-PK1 cells were infected or mock-infected. Thirty minutes prior to fixation, cells were incubated with 2 mM 5-Bromouridine (BrU) and 15 μM actinomycin D. Cells were fixed and labelled with anti-BrdU (green) and either anti-N or anti-ER antibodies (red). Nuclei were stained with DAPI (blue), scale bar indicates 10 µm.

    Techniques Used: Infection, Incubation, Staining

    PDCoV-associated dsRNA can be detected from 4 h post-infection (hpi). LLC-PK1 cells were infected or mock-infected. At the stated timepoints, cells were fixed and labelled with anti-dsRNA (green) and anti-N (red). Nuclei were stained with DAPI (blue) and scale bar indicates 10 μm. Images are representative of three independent replicates.
    Figure Legend Snippet: PDCoV-associated dsRNA can be detected from 4 h post-infection (hpi). LLC-PK1 cells were infected or mock-infected. At the stated timepoints, cells were fixed and labelled with anti-dsRNA (green) and anti-N (red). Nuclei were stained with DAPI (blue) and scale bar indicates 10 μm. Images are representative of three independent replicates.

    Techniques Used: Infection, Staining

    PDCoV RNA synthesis was detected from 3 hpi. LLC-PK1 cells were infected or mock-infected. Thirty minutes prior to the indicated fixation time, cells were incubated with 2 mM 5-Bromouridine (BrU) and 15 µM actinomycin D (ActD). Mock cells were incubated with (+ActD) and without (−ActD) actinomycin D as indicated. Cells were fixed and RNA containing BrU were detected using an anti-BrdU antibody (green). Nuclei were stained with DAPI (blue), scale bar indicates 10 μm. Images representative of three independent replicates.
    Figure Legend Snippet: PDCoV RNA synthesis was detected from 3 hpi. LLC-PK1 cells were infected or mock-infected. Thirty minutes prior to the indicated fixation time, cells were incubated with 2 mM 5-Bromouridine (BrU) and 15 µM actinomycin D (ActD). Mock cells were incubated with (+ActD) and without (−ActD) actinomycin D as indicated. Cells were fixed and RNA containing BrU were detected using an anti-BrdU antibody (green). Nuclei were stained with DAPI (blue), scale bar indicates 10 μm. Images representative of three independent replicates.

    Techniques Used: Infection, Incubation, Staining

    PDCoV RO is made up of double-membrane vesicles (DMVs) and zippered ER with double-membrane spherules. LLC-PK1 cells were mock-infected ( A ) or infected with PDCoV ( B – F ). At 8 hpi, cells were fixed with glutaraldehyde and processed for transmission electron microscopy. Virions in vesicles are indicated with black arrows, DMVs are indicated with white arrows, and regions of zippered ER with spherules are indicated with black brackets. Scale bars indicate 1 µm ( A , B ) or 500 nm ( C – F ).
    Figure Legend Snippet: PDCoV RO is made up of double-membrane vesicles (DMVs) and zippered ER with double-membrane spherules. LLC-PK1 cells were mock-infected ( A ) or infected with PDCoV ( B – F ). At 8 hpi, cells were fixed with glutaraldehyde and processed for transmission electron microscopy. Virions in vesicles are indicated with black arrows, DMVs are indicated with white arrows, and regions of zippered ER with spherules are indicated with black brackets. Scale bars indicate 1 µm ( A , B ) or 500 nm ( C – F ).

    Techniques Used: Infection, Transmission Assay, Electron Microscopy

    Dynamics of porcine deltacoronavirus (PDCoV) OH-FD22 replication in LLC-PK1 cells. ( A ) LLC-PK1 cells were infected with PDCoV and at the indicated timepoints, RNA was harvested and reverse transcribed to cDNA. The copy number of cDNA was quantified by quantitative polymerase chain reaction (qPCR) using a standard curve and normalized to mock-infected cells. Mean and standard deviation of three independent replicates are shown. ( B ) LLC-PK1 cells were PDCoV-infected or mock-infected. Total cell lysate was harvested at the stated timepoints and viral nucleoprotein detected (Anti-N) by western blot. Actin (Anti-actin) was used as a loading control. Molecular weight markers are shown. Blot representative of three independent repeats. ( C ) LLC-PK1 cells were infected with PDCoV and at the indicated timepoints and cell culture media was harvested. The titer of progeny virus was determined by tissue culture infectious dose 50 (TCID 50 ). Mean and standard deviation from three independent replicates are shown.
    Figure Legend Snippet: Dynamics of porcine deltacoronavirus (PDCoV) OH-FD22 replication in LLC-PK1 cells. ( A ) LLC-PK1 cells were infected with PDCoV and at the indicated timepoints, RNA was harvested and reverse transcribed to cDNA. The copy number of cDNA was quantified by quantitative polymerase chain reaction (qPCR) using a standard curve and normalized to mock-infected cells. Mean and standard deviation of three independent replicates are shown. ( B ) LLC-PK1 cells were PDCoV-infected or mock-infected. Total cell lysate was harvested at the stated timepoints and viral nucleoprotein detected (Anti-N) by western blot. Actin (Anti-actin) was used as a loading control. Molecular weight markers are shown. Blot representative of three independent repeats. ( C ) LLC-PK1 cells were infected with PDCoV and at the indicated timepoints and cell culture media was harvested. The titer of progeny virus was determined by tissue culture infectious dose 50 (TCID 50 ). Mean and standard deviation from three independent replicates are shown.

    Techniques Used: Infection, Real-time Polymerase Chain Reaction, Standard Deviation, Western Blot, Molecular Weight, Cell Culture

    DMVs and zippered ER with double-membrane spherules were present from 6 hpi to 24 hpi. LLC-PK1 cells were infected with PDCoV, and at 6 hpi ( A , B ) or 24 hpi ( C , D ), cells were fixed with glutaraldehyde and processed for electron microscopy. Virions in vesicles are indicated with black arrows, DMVs are indicated with white arrows, and regions of zippered ER with spherules are indicated with black brackets. Scale bars indicate 200 nm ( A ) or 500 nm ( B – D ).
    Figure Legend Snippet: DMVs and zippered ER with double-membrane spherules were present from 6 hpi to 24 hpi. LLC-PK1 cells were infected with PDCoV, and at 6 hpi ( A , B ) or 24 hpi ( C , D ), cells were fixed with glutaraldehyde and processed for electron microscopy. Virions in vesicles are indicated with black arrows, DMVs are indicated with white arrows, and regions of zippered ER with spherules are indicated with black brackets. Scale bars indicate 200 nm ( A ) or 500 nm ( B – D ).

    Techniques Used: Infection, Electron Microscopy

    9) Product Images from "Increase in Protective Effect of Panax vietnamensis by Heat Processing on Cisplatin-Induced Kidney Cell Toxicity"

    Article Title: Increase in Protective Effect of Panax vietnamensis by Heat Processing on Cisplatin-Induced Kidney Cell Toxicity

    Journal: Molecules

    doi: 10.3390/molecules24244627

    Kidney cell protective effect of PVG fractions against cisplatin toxicity on LLC-PK1 cells. Results are expressed as mean ± SD (n = 3), * p
    Figure Legend Snippet: Kidney cell protective effect of PVG fractions against cisplatin toxicity on LLC-PK1 cells. Results are expressed as mean ± SD (n = 3), * p

    Techniques Used:

    Scheme of the activity-guided fractionation and isolation of potential kidney cell protective compounds from PVG fractions against cisplatin toxicity on LLC-PK1 cells.
    Figure Legend Snippet: Scheme of the activity-guided fractionation and isolation of potential kidney cell protective compounds from PVG fractions against cisplatin toxicity on LLC-PK1 cells.

    Techniques Used: Activity Assay, Fractionation, Isolation

    10) Product Images from "Palladin is Upregulated in Kidney Disease and Contributes to Epithelial Cell Migration After Injury"

    Article Title: Palladin is Upregulated in Kidney Disease and Contributes to Epithelial Cell Migration After Injury

    Journal: Scientific Reports

    doi: 10.1038/srep07695

    Palladin expression in α-MPO model of ANCA and human kidney diseases. IHC staining of mouse kidney cortex with 622 and PALL75 (A) show an increase in palladin expression in tubules and crescents as disease progresses from 4 days up to 12 days after disease induction. Sections from medulla show an increase in staining in the vasa recta of the medulla with intensity parallel to disease severity (B). Palladin expression in human biopsy specimens. IHC staining of biopsy sections from different human kidney diseases with PALL75 (C). Arrows denote podocytes; arrowheads denote proximal tubule cells; double-headed arrows denote distal tubule cells; (*) denote smooth muscle cells; C denotes crescents. IHC staining of three patients with non-immunologic mediated kidney injury and ATN with PALL75 (D).
    Figure Legend Snippet: Palladin expression in α-MPO model of ANCA and human kidney diseases. IHC staining of mouse kidney cortex with 622 and PALL75 (A) show an increase in palladin expression in tubules and crescents as disease progresses from 4 days up to 12 days after disease induction. Sections from medulla show an increase in staining in the vasa recta of the medulla with intensity parallel to disease severity (B). Palladin expression in human biopsy specimens. IHC staining of biopsy sections from different human kidney diseases with PALL75 (C). Arrows denote podocytes; arrowheads denote proximal tubule cells; double-headed arrows denote distal tubule cells; (*) denote smooth muscle cells; C denotes crescents. IHC staining of three patients with non-immunologic mediated kidney injury and ATN with PALL75 (D).

    Techniques Used: Expressing, Immunohistochemistry, Staining

    Validation of PALL75 and palladin isoforms in mouse and human tissue. PALL75 recognizes predominantly a band ~75 kDa which is knocked down in stable knockdown lines from human cancer-associated fibroblasts, on which we have previously published and designated shRNA1 and shRNA2 16 . A less intense band at ~130 kDa is also present, which probably represents isoform 3. Although predicted MW of isoform 3 is ~108 kDa, it actually runs at ~130 kDa as confirmed by mass spectrometry of excised 130 kDa band in a human cell line (data not shown). GAPDH shown for loading control (A). Different isoforms in mouse and human kidney. Proteins extracted from kidneys of wild type mice and human were immunoblotted with antibodies to palladin. One representative sample (B). 621 detects isoform 4 most robustly. Other bands are present which may represent lower-prevalence variants. PALL75 detects isoform 4 most robustly. Immunohistochemical staining of C57BL/6 wild type mouse kidney with 622 (C, D) and PALL75 (E, F). Vessels are shown with asterisks (*)as palladin is known to be present in smooth muscle cells. Arrows denote podocytes. Arrowheads denote proximal tubule cells. No primary antibody control (G).
    Figure Legend Snippet: Validation of PALL75 and palladin isoforms in mouse and human tissue. PALL75 recognizes predominantly a band ~75 kDa which is knocked down in stable knockdown lines from human cancer-associated fibroblasts, on which we have previously published and designated shRNA1 and shRNA2 16 . A less intense band at ~130 kDa is also present, which probably represents isoform 3. Although predicted MW of isoform 3 is ~108 kDa, it actually runs at ~130 kDa as confirmed by mass spectrometry of excised 130 kDa band in a human cell line (data not shown). GAPDH shown for loading control (A). Different isoforms in mouse and human kidney. Proteins extracted from kidneys of wild type mice and human were immunoblotted with antibodies to palladin. One representative sample (B). 621 detects isoform 4 most robustly. Other bands are present which may represent lower-prevalence variants. PALL75 detects isoform 4 most robustly. Immunohistochemical staining of C57BL/6 wild type mouse kidney with 622 (C, D) and PALL75 (E, F). Vessels are shown with asterisks (*)as palladin is known to be present in smooth muscle cells. Arrows denote podocytes. Arrowheads denote proximal tubule cells. No primary antibody control (G).

    Techniques Used: Mass Spectrometry, Mouse Assay, Immunohistochemistry, Staining

    11) Product Images from "CD163 and pAPN double-knockout pigs are resistant to PRRSV and TGEV and exhibit decreased susceptibility to PDCoV while maintaining normal production performance"

    Article Title: CD163 and pAPN double-knockout pigs are resistant to PRRSV and TGEV and exhibit decreased susceptibility to PDCoV while maintaining normal production performance

    Journal: eLife

    doi: 10.7554/eLife.57132

    PAMs of DKO pigs exhibit reduced susceptibility to PDCoV. ( A ) WT PAMs and DKO PAMs were infected or mock-infected with PDCoV (MOI = 10); at 24 hpi, PDCoV-N-specific fluorescence signals were detected by IFA. ( B ) WT PAMs and DKO PAMs were infected with PDCoV (MOI = 10); at 24 hpi, cells were collected and the viral titer was determined by TCID 50 assays (in LLC-PK1 cells). ( C, D ) WT PAMs and DKO PAMs were infected with PDCoV (MOI = 10); at 24 hpi, cells were harvested and analyzed using qRT-PCR ( C ) and western blot assays ( D ). The PDCoV-N protein level was quantified by ImageJ. Data are expressed as the mean ± SD. Statistical significance was determined by Student’s t test; ns, p > 0.05; *p
    Figure Legend Snippet: PAMs of DKO pigs exhibit reduced susceptibility to PDCoV. ( A ) WT PAMs and DKO PAMs were infected or mock-infected with PDCoV (MOI = 10); at 24 hpi, PDCoV-N-specific fluorescence signals were detected by IFA. ( B ) WT PAMs and DKO PAMs were infected with PDCoV (MOI = 10); at 24 hpi, cells were collected and the viral titer was determined by TCID 50 assays (in LLC-PK1 cells). ( C, D ) WT PAMs and DKO PAMs were infected with PDCoV (MOI = 10); at 24 hpi, cells were harvested and analyzed using qRT-PCR ( C ) and western blot assays ( D ). The PDCoV-N protein level was quantified by ImageJ. Data are expressed as the mean ± SD. Statistical significance was determined by Student’s t test; ns, p > 0.05; *p

    Techniques Used: Infection, Fluorescence, Immunofluorescence, Quantitative RT-PCR, Western Blot

    12) Product Images from "Chemical Characterization of a Renoprotective Metabolite from Termite-Associated Streptomyces sp. RB1 against Cisplatin-Induced Cytotoxicity"

    Article Title: Chemical Characterization of a Renoprotective Metabolite from Termite-Associated Streptomyces sp. RB1 against Cisplatin-Induced Cytotoxicity

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms19010174

    Protective effect of ABR on apoptosis in LLC-PK1 cells exposed to 25 μM cisplatin for 24 h by western blot ( A ) Expression levels of MAPK-caspase-3 pathway proteins; ( B ) Each bar graph represents densitometric quantification of western blot bands. Control cells were treated with the vehicle only (mean ± SD, * p
    Figure Legend Snippet: Protective effect of ABR on apoptosis in LLC-PK1 cells exposed to 25 μM cisplatin for 24 h by western blot ( A ) Expression levels of MAPK-caspase-3 pathway proteins; ( B ) Each bar graph represents densitometric quantification of western blot bands. Control cells were treated with the vehicle only (mean ± SD, * p

    Techniques Used: Western Blot, Expressing

    Protective effect of ABR on apoptosis in LLC-PK1 cells exposed to 25 μM cisplatin for 24 h by image-based cytometric assay. ( A ) Representative images for apoptosis detection (green) (Magnification: 4×); ( B ) Percentage of Annexin V-positive-stained apoptotic cells. Control cells were treated with the vehicle only (mean ± SD, * p
    Figure Legend Snippet: Protective effect of ABR on apoptosis in LLC-PK1 cells exposed to 25 μM cisplatin for 24 h by image-based cytometric assay. ( A ) Representative images for apoptosis detection (green) (Magnification: 4×); ( B ) Percentage of Annexin V-positive-stained apoptotic cells. Control cells were treated with the vehicle only (mean ± SD, * p

    Techniques Used: Staining

    Protective effect of 1- O -(2-aminobenzoyl)-α- l -rhamnopyranoside (ABR) in LLC-PK1 cells exposed to 25 μM of cisplatin for 24 h by MTT assay. ( A ) Chemical structure of ABR. Dose-dependent protective effects of ( B ) ABR and ( C ) N -acetyl cysteine (NAC) in LLC-PK1 cells exposed to 25 μM of cisplatin for 24 h by MTT assay; ( D ) Combined effects of ABR and cisplatin on the cell viability in human ovarian cancer A2780 cells. NAC was used as a positive control. Control cells were treated with the vehicle only (mean ± SD, * p
    Figure Legend Snippet: Protective effect of 1- O -(2-aminobenzoyl)-α- l -rhamnopyranoside (ABR) in LLC-PK1 cells exposed to 25 μM of cisplatin for 24 h by MTT assay. ( A ) Chemical structure of ABR. Dose-dependent protective effects of ( B ) ABR and ( C ) N -acetyl cysteine (NAC) in LLC-PK1 cells exposed to 25 μM of cisplatin for 24 h by MTT assay; ( D ) Combined effects of ABR and cisplatin on the cell viability in human ovarian cancer A2780 cells. NAC was used as a positive control. Control cells were treated with the vehicle only (mean ± SD, * p

    Techniques Used: MTT Assay, Positive Control

    Protective effects of the MeOH extract (ME) of Streptomyces sp. RB1 and its fractions (hexane-soluble (H), CH 2 Cl 2 -soluble (C), EtOAc-soluble (EA), and n -BuOH-soluble fractions (BU)) in LLC-PK1 cells exposed to 25 μM of cisplatin for 24 h by MTT assay. Protective effects of ( A ) ME, and ( B ) H fraction, ( C ) C fraction, ( D ) EA fraction, and ( E ) BU fraction in LLC-PK1 cells exposed to 25 μM of cisplatin for 24 h by MTT assay. The concentrations of samples were 0, 10, 50, 100 and 250 μg/mL. Control cells were treated with the vehicle only (mean ± SD, * p
    Figure Legend Snippet: Protective effects of the MeOH extract (ME) of Streptomyces sp. RB1 and its fractions (hexane-soluble (H), CH 2 Cl 2 -soluble (C), EtOAc-soluble (EA), and n -BuOH-soluble fractions (BU)) in LLC-PK1 cells exposed to 25 μM of cisplatin for 24 h by MTT assay. Protective effects of ( A ) ME, and ( B ) H fraction, ( C ) C fraction, ( D ) EA fraction, and ( E ) BU fraction in LLC-PK1 cells exposed to 25 μM of cisplatin for 24 h by MTT assay. The concentrations of samples were 0, 10, 50, 100 and 250 μg/mL. Control cells were treated with the vehicle only (mean ± SD, * p

    Techniques Used: MTT Assay

    13) Product Images from "Myocardin-related transcription factor and serum response factor regulate cilium turnover by both transcriptional and local mechanisms"

    Article Title: Myocardin-related transcription factor and serum response factor regulate cilium turnover by both transcriptional and local mechanisms

    Journal: iScience

    doi: 10.1016/j.isci.2021.102739

    MRTF and SRF localize to the basal body and the primary cilium (A) LLC-PK1 (left panels) and RPE cells (right panels) were serum-deprived and co-stained for MRTF and the basal body marker γ-tubulin. Staining was visualized by confocal microscopy. Scale bar 20 μm and 5 μm for full and boxed images, respectively. The last panels (Box) show the magnified regions demarcated by the dashed rectangles in the corresponding merged images. Note that distinct MRTF patches colocalize or partially overlap with γ-tubulin (BB), but MRTF staining occasionally penetrates the cilium as well. (B) LLC-PK1 cells (left panels) and RPE cells (right panels) were co-stained for SRF and the cilium marker acetylated tubulin. The last panels (Box) show the magnified regions demarcated by the dashed rectangles in the corresponding merged images. Note that SRF, as expected, resides primarily in the nucleus. In addition, SRF also shows clear localization to the primary cilium in a subset of cells.
    Figure Legend Snippet: MRTF and SRF localize to the basal body and the primary cilium (A) LLC-PK1 (left panels) and RPE cells (right panels) were serum-deprived and co-stained for MRTF and the basal body marker γ-tubulin. Staining was visualized by confocal microscopy. Scale bar 20 μm and 5 μm for full and boxed images, respectively. The last panels (Box) show the magnified regions demarcated by the dashed rectangles in the corresponding merged images. Note that distinct MRTF patches colocalize or partially overlap with γ-tubulin (BB), but MRTF staining occasionally penetrates the cilium as well. (B) LLC-PK1 cells (left panels) and RPE cells (right panels) were co-stained for SRF and the cilium marker acetylated tubulin. The last panels (Box) show the magnified regions demarcated by the dashed rectangles in the corresponding merged images. Note that SRF, as expected, resides primarily in the nucleus. In addition, SRF also shows clear localization to the primary cilium in a subset of cells.

    Techniques Used: Staining, Marker, Confocal Microscopy

    MRTF and SRF are present in isolated cilia and interact with the components of the deciliation machinery (A) Primary cilia were isolated from LLC-PK1 cells as described in STAR Methods . Left panel: An aliquot of the preparation was layered onto a coverslip, fixed, stained with acetylated tubulin, and visualized by immunofluorescence microscopy. Note the compact tubular and vesicular acetylated tubulin-positive structures, indicating large enrichment of PC-derived material. Right panel: Quality control of the cilium preparation. Equal amount of protein (5 μg) from cilium preparations and the non-ciliary fraction (residual whole cell lysates) of serum-deprived cells were analyzed by Western blotting using ciliary (Ac-Tub, Arl13b), nuclear (histone), nuclear/cytosolic (Nup98) and cytosolic (GAPDH) markers. Note the robust enrichment of ciliary markers and the absence of the nuclear marker in the cilium preparation. (B) The effect of serum stimulation on the level of indicated proteins in PC preparations as determined by Western blotting. LLC-PK1 cells were serum-deprived for 48 hr, and then left untreated or exposed to 10% serum-containing medium for 2 hr. Note that serum induces substantial recruitment of MRTF and increases SRF phosphorylation in the PC. (C) LLC-PK cells were transfected with siNR or siMRTF for 24 hr, serum-deprived for 48 hr, and then incubated for 3h in the absence or presence of serum. Primary cilia were then isolated from each cell population. The ciliary content of the indicated proteins (Input) is shown in the left panel. These preparations were then subjected to immunoprecipitation with an anti-SRF antibody (IP SRF), and the precipitates were probed for SRF and the indicated proteins, shown in the right panel. (D–I) Quantification of the indicated proteins in cilium fractions isolated from control (NR) or MRTF-silenced (MRTF) cells, under serum-free (SF) or serum-stimulated (Ser) conditions. The average density obtained from the control fractions (siNR-transfected cells, SF condition) was set to 1 for each protein, and means ± SD are shown. ∗∗∗p
    Figure Legend Snippet: MRTF and SRF are present in isolated cilia and interact with the components of the deciliation machinery (A) Primary cilia were isolated from LLC-PK1 cells as described in STAR Methods . Left panel: An aliquot of the preparation was layered onto a coverslip, fixed, stained with acetylated tubulin, and visualized by immunofluorescence microscopy. Note the compact tubular and vesicular acetylated tubulin-positive structures, indicating large enrichment of PC-derived material. Right panel: Quality control of the cilium preparation. Equal amount of protein (5 μg) from cilium preparations and the non-ciliary fraction (residual whole cell lysates) of serum-deprived cells were analyzed by Western blotting using ciliary (Ac-Tub, Arl13b), nuclear (histone), nuclear/cytosolic (Nup98) and cytosolic (GAPDH) markers. Note the robust enrichment of ciliary markers and the absence of the nuclear marker in the cilium preparation. (B) The effect of serum stimulation on the level of indicated proteins in PC preparations as determined by Western blotting. LLC-PK1 cells were serum-deprived for 48 hr, and then left untreated or exposed to 10% serum-containing medium for 2 hr. Note that serum induces substantial recruitment of MRTF and increases SRF phosphorylation in the PC. (C) LLC-PK cells were transfected with siNR or siMRTF for 24 hr, serum-deprived for 48 hr, and then incubated for 3h in the absence or presence of serum. Primary cilia were then isolated from each cell population. The ciliary content of the indicated proteins (Input) is shown in the left panel. These preparations were then subjected to immunoprecipitation with an anti-SRF antibody (IP SRF), and the precipitates were probed for SRF and the indicated proteins, shown in the right panel. (D–I) Quantification of the indicated proteins in cilium fractions isolated from control (NR) or MRTF-silenced (MRTF) cells, under serum-free (SF) or serum-stimulated (Ser) conditions. The average density obtained from the control fractions (siNR-transfected cells, SF condition) was set to 1 for each protein, and means ± SD are shown. ∗∗∗p

    Techniques Used: Isolation, Staining, Immunofluorescence, Microscopy, Derivative Assay, Western Blot, Marker, Transfection, Incubation, Immunoprecipitation

    MRTF interacts with AurA and inhibits its degradation; non-transcriptional effects of MRTF (A) LLC-PK1 cells were transfected with the indicated siRNAs, and 24 hr later placed in serum-free medium supplemented with vehicle (DMSO) or the proteasome inhibitor MG-132 (20 μM) as indicated. After an additional 24 hr, cell lysates were prepared for Western blotting and probed for the indicated proteins. (B) Experiments as in A were quantified by densitometry, and results were normalized to the level of NR-transfected DMSO-treated control is each experiment (n = 3). ∗p
    Figure Legend Snippet: MRTF interacts with AurA and inhibits its degradation; non-transcriptional effects of MRTF (A) LLC-PK1 cells were transfected with the indicated siRNAs, and 24 hr later placed in serum-free medium supplemented with vehicle (DMSO) or the proteasome inhibitor MG-132 (20 μM) as indicated. After an additional 24 hr, cell lysates were prepared for Western blotting and probed for the indicated proteins. (B) Experiments as in A were quantified by densitometry, and results were normalized to the level of NR-transfected DMSO-treated control is each experiment (n = 3). ∗p

    Techniques Used: Transfection, Western Blot

    MRTF and SRF are necessary for serum-induced primary cilium resorption (A) LLC-PK1 cells were transfected with control (non-related, NR) or specific siRNAs against pig MRTF A/B (siMRTF) or SRF (siSRF) (50 nM for each gene) for 24 hr under serum-free conditions. The cells were then exposed to fresh serum-free or 10% serum-containing medium (serum) for 24 hr. Primary cilia were visualized by acetylated tubulin staining (red) and nuclei by Dapi (blue). Bar: 20 μm. (B) Effective silencing of MRTF and SRF in LLC-PK1 cells was verified by immunoblotting. (C) Time course of serum-induced primary cilium resorption in LLC-PK1 cells transfected with NR, siMRTF or siSRF. The percentage of ciliated cells was determined using acetylated tubulin staining for each time point (0, 4, 8, 12, 24 hr); n = 5–6 for each time point, with ≈150 cells counted/condition. The plot shows each individual point, and the mean ± SEM. The percentage of ciliated NR-transfected cells was compared to that of siMRTF- or siSRF-transfected cells at each corresponding time point using unpaired t test. ∗p
    Figure Legend Snippet: MRTF and SRF are necessary for serum-induced primary cilium resorption (A) LLC-PK1 cells were transfected with control (non-related, NR) or specific siRNAs against pig MRTF A/B (siMRTF) or SRF (siSRF) (50 nM for each gene) for 24 hr under serum-free conditions. The cells were then exposed to fresh serum-free or 10% serum-containing medium (serum) for 24 hr. Primary cilia were visualized by acetylated tubulin staining (red) and nuclei by Dapi (blue). Bar: 20 μm. (B) Effective silencing of MRTF and SRF in LLC-PK1 cells was verified by immunoblotting. (C) Time course of serum-induced primary cilium resorption in LLC-PK1 cells transfected with NR, siMRTF or siSRF. The percentage of ciliated cells was determined using acetylated tubulin staining for each time point (0, 4, 8, 12, 24 hr); n = 5–6 for each time point, with ≈150 cells counted/condition. The plot shows each individual point, and the mean ± SEM. The percentage of ciliated NR-transfected cells was compared to that of siMRTF- or siSRF-transfected cells at each corresponding time point using unpaired t test. ∗p

    Techniques Used: Transfection, Staining

    MRTF silencing promotes ciliogenesis even in the presence of serum, partially via a transcription-independent mechanism (A and B) LLC-PK1 cells were transfected with siNR or siMRTF and continuously kept in serum-containing medium for 48 hr before processing for immunofluorescence. Cells were stained with primary cilia markers Acetylated tubulin (A) or Arl13b (B). Scale bar: 20 μm. (C) The percentage of ciliated cells was determined using both cilium markers (mean ± SD, n = 6, ≈200 cells/condition). ∗∗∗p
    Figure Legend Snippet: MRTF silencing promotes ciliogenesis even in the presence of serum, partially via a transcription-independent mechanism (A and B) LLC-PK1 cells were transfected with siNR or siMRTF and continuously kept in serum-containing medium for 48 hr before processing for immunofluorescence. Cells were stained with primary cilia markers Acetylated tubulin (A) or Arl13b (B). Scale bar: 20 μm. (C) The percentage of ciliated cells was determined using both cilium markers (mean ± SD, n = 6, ≈200 cells/condition). ∗∗∗p

    Techniques Used: Transfection, Immunofluorescence, Staining

    14) Product Images from "Ergosterol peroxide exhibits antiviral and immunomodulatory abilities against porcine deltacoronavirus (PDCoV) via suppression of NF-κB and p38/MAPK signaling pathways in vitro"

    Article Title: Ergosterol peroxide exhibits antiviral and immunomodulatory abilities against porcine deltacoronavirus (PDCoV) via suppression of NF-κB and p38/MAPK signaling pathways in vitro

    Journal: International Immunopharmacology

    doi: 10.1016/j.intimp.2020.107317

    Ergosterol peroxide alleviates PDCoV-induced mRNA expressions of cytokines in LLC-PK1 cells. LLC-PK1 cells were inoculated with PDCoV (MOI=0.5) or transfected with polyI:C in the presence or absence of EP. Total RNA was extracted from cell lysates at 24 hpi. The relative expression of IL-1β mRNA (A), IL-6 mRNA (B), IL-12 mRNA (C), TNF-α mRNA (D), IFN-α mRNA (E), IFN-β mRNA (F), Mx1 mRNA (G), OAS mRNA (H) and PKR mRNA (I) was assessed by RT-qPCR. Values represent the mean±SD for three independent experiments. * P
    Figure Legend Snippet: Ergosterol peroxide alleviates PDCoV-induced mRNA expressions of cytokines in LLC-PK1 cells. LLC-PK1 cells were inoculated with PDCoV (MOI=0.5) or transfected with polyI:C in the presence or absence of EP. Total RNA was extracted from cell lysates at 24 hpi. The relative expression of IL-1β mRNA (A), IL-6 mRNA (B), IL-12 mRNA (C), TNF-α mRNA (D), IFN-α mRNA (E), IFN-β mRNA (F), Mx1 mRNA (G), OAS mRNA (H) and PKR mRNA (I) was assessed by RT-qPCR. Values represent the mean±SD for three independent experiments. * P

    Techniques Used: Transfection, Expressing, Quantitative RT-PCR

    Ergosterol peroxide directly inactivates PDCoV virions. The viricidal effect of EP on PDCoV was determined by RT-qPCR (A), viral titer (lgTCID 50 /mL) (B) and IFA (C). The effect of preincubation of cells with EP for 2 h on PDCoV infection was determined by RT-qPCR (E), viral titer (lgTCID 50 /ml) (F) and IFA (G). D, H. Percentage of infected cells in different treatment groups in C and G. RT-qPCR was performed with primers targeting the PDCoV S gene. Viral titer (lgTCID50/mL) in LLC-PK1 supernatant was calculated by the method of Reed and Muench. Green fluorescence represents the PDCoV distribution, and the blue fluorescence represents the nuclear distribution. Values represent the mean ± SD for three independent experiments. * P
    Figure Legend Snippet: Ergosterol peroxide directly inactivates PDCoV virions. The viricidal effect of EP on PDCoV was determined by RT-qPCR (A), viral titer (lgTCID 50 /mL) (B) and IFA (C). The effect of preincubation of cells with EP for 2 h on PDCoV infection was determined by RT-qPCR (E), viral titer (lgTCID 50 /ml) (F) and IFA (G). D, H. Percentage of infected cells in different treatment groups in C and G. RT-qPCR was performed with primers targeting the PDCoV S gene. Viral titer (lgTCID50/mL) in LLC-PK1 supernatant was calculated by the method of Reed and Muench. Green fluorescence represents the PDCoV distribution, and the blue fluorescence represents the nuclear distribution. Values represent the mean ± SD for three independent experiments. * P

    Techniques Used: Quantitative RT-PCR, Immunofluorescence, Infection, Fluorescence

    The cytotoxicity of ergosterol peroxide on LLC-PK1 cells. A. Chemical structure of EP. B. Determination of cytotoxicity of EP by CCK8 assay. Cells were treated with 0, 3.9, 7.8, 15.6, 31, 62, 124 and 248 μM EP for 36 h, respectively. The relative cell viability was evaluated by CCK8 Kit according to the manufacturer’s instructions. Values represent the mean ± SD for three independent experiments. * P
    Figure Legend Snippet: The cytotoxicity of ergosterol peroxide on LLC-PK1 cells. A. Chemical structure of EP. B. Determination of cytotoxicity of EP by CCK8 assay. Cells were treated with 0, 3.9, 7.8, 15.6, 31, 62, 124 and 248 μM EP for 36 h, respectively. The relative cell viability was evaluated by CCK8 Kit according to the manufacturer’s instructions. Values represent the mean ± SD for three independent experiments. * P

    Techniques Used: CCK-8 Assay

    Antiviral effect of ergosterol peroxide on different stages of PDCoV life cycle. The viral RNA copies in LLC-PK1 cells treated with various concentrations of EP during viral attachment (A), entry (B) and post-entry stage (C) were determined by RT-qPCR with primers targeting the PDCoV S gene. D. The viral titer (lgTCID 50 /mL) in LLC-PK1 supernatants treated with different concentrations of EP in the post-entry stage was calculated by the method of Reed and Muench. E. Inhibitory effects of EP on PDCoV in the post-entry stage were observed by IFA. Green fluorescence represents the PDCoV distribution, and the blue fluorescence represents the nuclear distribution. F. Percentage of infected cells in different treatment groups in E. Values represent the mean ± SD for three independent experiments. * P
    Figure Legend Snippet: Antiviral effect of ergosterol peroxide on different stages of PDCoV life cycle. The viral RNA copies in LLC-PK1 cells treated with various concentrations of EP during viral attachment (A), entry (B) and post-entry stage (C) were determined by RT-qPCR with primers targeting the PDCoV S gene. D. The viral titer (lgTCID 50 /mL) in LLC-PK1 supernatants treated with different concentrations of EP in the post-entry stage was calculated by the method of Reed and Muench. E. Inhibitory effects of EP on PDCoV in the post-entry stage were observed by IFA. Green fluorescence represents the PDCoV distribution, and the blue fluorescence represents the nuclear distribution. F. Percentage of infected cells in different treatment groups in E. Values represent the mean ± SD for three independent experiments. * P

    Techniques Used: Quantitative RT-PCR, Immunofluorescence, Fluorescence, Infection

    Antiviral effect of ergosterol peroxide on PDCoV replication. A. The protein levels of PDCoV N in LLC-PK1cells were determined by Western blotting. B. Results were presented as the ratio of protein band intensity to the intensity of the β-actin band. C. The viral RNA copies in LLC-PK1 cells were determined by RT-qPCR with primers targeting the PDCoV S gene. D. The viral titer (lgTCID 50 /mL) in LLC-PK1 supernatants was calculated by the method of Reed and Muench. E. PDCoV replication in LLC-PK1 cells was determined by IFA. Green fluorescence represents the PDCoV distribution, and the blue fluorescence represents the nuclear distribution. F. Percentage of infected cells in different treatment groups in E. Values represent the mean ± SD for three independent experiments. * P
    Figure Legend Snippet: Antiviral effect of ergosterol peroxide on PDCoV replication. A. The protein levels of PDCoV N in LLC-PK1cells were determined by Western blotting. B. Results were presented as the ratio of protein band intensity to the intensity of the β-actin band. C. The viral RNA copies in LLC-PK1 cells were determined by RT-qPCR with primers targeting the PDCoV S gene. D. The viral titer (lgTCID 50 /mL) in LLC-PK1 supernatants was calculated by the method of Reed and Muench. E. PDCoV replication in LLC-PK1 cells was determined by IFA. Green fluorescence represents the PDCoV distribution, and the blue fluorescence represents the nuclear distribution. F. Percentage of infected cells in different treatment groups in E. Values represent the mean ± SD for three independent experiments. * P

    Techniques Used: Western Blot, Quantitative RT-PCR, Immunofluorescence, Fluorescence, Infection

    The IκBα and p38/MAPK signaling pathways might be involved in the anti-PDCoV mechanisms of EP in LLC-PK1 cells. A. LLC-PK1 cells were inoculated with PDCoV (MOI=0.5) or transfected with polyI:C in the presence or absence of EP. Western blotting analysis of p-IκBα and IκBα was determined at 24 hpi. B. LLC-PK1 cells were pretreated with different concentrations of p38 inhibitor SB203580 for 1 h, followed by PDCoV infection (MOI=0.1) in the presence or absence of EP. Western blotting analysis of p-p38, p38 and PDCoV N was determined at 24 hpi. C, D. Results were presented as the ratio of protein band intensity to the intensity of the β-actin band. Values represent the mean ± SD for three independent experiments. * P
    Figure Legend Snippet: The IκBα and p38/MAPK signaling pathways might be involved in the anti-PDCoV mechanisms of EP in LLC-PK1 cells. A. LLC-PK1 cells were inoculated with PDCoV (MOI=0.5) or transfected with polyI:C in the presence or absence of EP. Western blotting analysis of p-IκBα and IκBα was determined at 24 hpi. B. LLC-PK1 cells were pretreated with different concentrations of p38 inhibitor SB203580 for 1 h, followed by PDCoV infection (MOI=0.1) in the presence or absence of EP. Western blotting analysis of p-p38, p38 and PDCoV N was determined at 24 hpi. C, D. Results were presented as the ratio of protein band intensity to the intensity of the β-actin band. Values represent the mean ± SD for three independent experiments. * P

    Techniques Used: Transfection, Western Blot, Infection

    Ergosterol peroxide suppresses PDCoV-induced activations of IκBα and p38 in LLC-PK1 cells. LLC-PK1 cells were inoculated with PDCoV (MOI = 0.5) in the presence or absence of EP. Western blotting analysis of p-IκBα and IκBα (A), p-NF-κB p65 and NF-κB p65 (B), p-ERK and ERK(C), p-JNK and JNK (D), p-p38 and p38 (E) was determined at 24 hpi. F-J. Results were presented as the ratio of protein band intensity to the intensity of the β-actin band. Values represent the mean±SD for three independent experiments. * P
    Figure Legend Snippet: Ergosterol peroxide suppresses PDCoV-induced activations of IκBα and p38 in LLC-PK1 cells. LLC-PK1 cells were inoculated with PDCoV (MOI = 0.5) in the presence or absence of EP. Western blotting analysis of p-IκBα and IκBα (A), p-NF-κB p65 and NF-κB p65 (B), p-ERK and ERK(C), p-JNK and JNK (D), p-p38 and p38 (E) was determined at 24 hpi. F-J. Results were presented as the ratio of protein band intensity to the intensity of the β-actin band. Values represent the mean±SD for three independent experiments. * P

    Techniques Used: Western Blot

    15) Product Images from "myo-Inositol Oxygenase Overexpression Accentuates Generation of Reactive Oxygen Species and Exacerbates Cellular Injury following High Glucose Ambience"

    Article Title: myo-Inositol Oxygenase Overexpression Accentuates Generation of Reactive Oxygen Species and Exacerbates Cellular Injury following High Glucose Ambience

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M115.669952

    A and B , effect of MIOX overexpression on TGF-β1 expression and bioactivity under HG ambience in LLC-PK1 cells. No significant increase in the expression or bioactivity was observed with transfection of EV (pcDNA) under basal LG conditions ( columns
    Figure Legend Snippet: A and B , effect of MIOX overexpression on TGF-β1 expression and bioactivity under HG ambience in LLC-PK1 cells. No significant increase in the expression or bioactivity was observed with transfection of EV (pcDNA) under basal LG conditions ( columns

    Techniques Used: Over Expression, Expressing, Transfection

    Expression and activity of MIOX in LLC-PK1 cells following its overexpression in HG ambience. Western blotting analyses show a dose-dependent increased expression of MIOX in response to various d -glucose concentrations, whereas treatment with MIOX-specific
    Figure Legend Snippet: Expression and activity of MIOX in LLC-PK1 cells following its overexpression in HG ambience. Western blotting analyses show a dose-dependent increased expression of MIOX in response to various d -glucose concentrations, whereas treatment with MIOX-specific

    Techniques Used: Expressing, Activity Assay, Over Expression, Western Blot

    16) Product Images from "PEDV ORF3 Independently Regulates IκB Kinase β-Mediated NF-κB and IFN-β Promoter Activities"

    Article Title: PEDV ORF3 Independently Regulates IκB Kinase β-Mediated NF-κB and IFN-β Promoter Activities

    Journal: Pathogens

    doi: 10.3390/pathogens9050376

    ORF3 and IκB kinase β (IKBKB) interaction and IKBKB’s effect on porcine epidemic diarrhea virus (PEDV) virus replication. ( A ) Indirect immunofluorescence confocal microscopy showing co-localization of ORF3 and IKBKB proteins in VeroE6 (left panel) and LLC-PK1 cells (right panel) . The plasmid expressing IKBKB with Flag-tag at the C-terminus was co-transfected with pCAGGS_ORF3-Myc in VeroE6 and LLC-PK1 cells. Cells transfected with empty plasmid was used as mock transfection. At 24 hpt, cells were incubated with mouse anti-Myc and rabbit anti-Flag antibodies. Goat anti-rabbit IgG Alexa flour 488 and goat anti-mouse IgG Alexa flour 647 were used as secondary antibodies. Protein localization was analyzed by confocal microscopy. ( B ) The plasmids expressing ORF3-Myc/ORF3-Flag and IKBKB-Flag/IKBKB-Myc proteins were co-transfected into HEK293T cells. The protein complexes were immunoprecipitated using anti-Myc bead. The immunoprecipitated proteins were probed with rabbit anti-Flag and anti-Myc antibodies. ( C ) Growth kinetics of the PEDV AV12 _ORF3 in overexpressed IKBKB- and IKBKB-knockout VeroE6 cells. VeroE6 (WT) cells, in triplicate, were transfected with a plasmid expressing IKBKB (upper panel). Cells transfected with empty vector were used as mock transfection control. At 8 hpt, cells were infected with PEDV AV12 _ORF3 at MOI of 0.1. Wild type (WT) and IKBKB knock-out (IKBKB KO) VeroE6 cells (lower panel), in triplicate, were infected with PEDV AV12 _ORF3 at MOI of 0.1. The virus ( n = 3) from each group was harvested at 24 and 48 hpi and subjected to SFU for virus titration. Statistical analysis was performed by using the student t-test method. Error bars represent the means ± standard error of means of virus titers. *, p
    Figure Legend Snippet: ORF3 and IκB kinase β (IKBKB) interaction and IKBKB’s effect on porcine epidemic diarrhea virus (PEDV) virus replication. ( A ) Indirect immunofluorescence confocal microscopy showing co-localization of ORF3 and IKBKB proteins in VeroE6 (left panel) and LLC-PK1 cells (right panel) . The plasmid expressing IKBKB with Flag-tag at the C-terminus was co-transfected with pCAGGS_ORF3-Myc in VeroE6 and LLC-PK1 cells. Cells transfected with empty plasmid was used as mock transfection. At 24 hpt, cells were incubated with mouse anti-Myc and rabbit anti-Flag antibodies. Goat anti-rabbit IgG Alexa flour 488 and goat anti-mouse IgG Alexa flour 647 were used as secondary antibodies. Protein localization was analyzed by confocal microscopy. ( B ) The plasmids expressing ORF3-Myc/ORF3-Flag and IKBKB-Flag/IKBKB-Myc proteins were co-transfected into HEK293T cells. The protein complexes were immunoprecipitated using anti-Myc bead. The immunoprecipitated proteins were probed with rabbit anti-Flag and anti-Myc antibodies. ( C ) Growth kinetics of the PEDV AV12 _ORF3 in overexpressed IKBKB- and IKBKB-knockout VeroE6 cells. VeroE6 (WT) cells, in triplicate, were transfected with a plasmid expressing IKBKB (upper panel). Cells transfected with empty vector were used as mock transfection control. At 8 hpt, cells were infected with PEDV AV12 _ORF3 at MOI of 0.1. Wild type (WT) and IKBKB knock-out (IKBKB KO) VeroE6 cells (lower panel), in triplicate, were infected with PEDV AV12 _ORF3 at MOI of 0.1. The virus ( n = 3) from each group was harvested at 24 and 48 hpi and subjected to SFU for virus titration. Statistical analysis was performed by using the student t-test method. Error bars represent the means ± standard error of means of virus titers. *, p

    Techniques Used: Immunofluorescence, Confocal Microscopy, Plasmid Preparation, Expressing, FLAG-tag, Transfection, Incubation, Immunoprecipitation, Knock-Out, Infection, Titration

    17) Product Images from "Correlative microscopy of freeze-dried cells and studies on intracellular calcium stores with imaging secondary ion mass spectrometry (SIMS)"

    Article Title: Correlative microscopy of freeze-dried cells and studies on intracellular calcium stores with imaging secondary ion mass spectrometry (SIMS)

    Journal: Journal of analytical atomic spectrometry

    doi: 10.1039/c9ja00193j

    SIMS images of 44 Ca influx and endogenous 40 Ca calcium in resting and stimulated renal epithelial LLC-PK1 cells. The cells were exposed for 5 minutes to a buffer containing 1.8 mM of the stable isotope 44 Ca in the presence of 1 µM of the calcium ionophore A23187 (middle row) and 100 nM of the arginine vasopressin hormone (bottom row).
    Figure Legend Snippet: SIMS images of 44 Ca influx and endogenous 40 Ca calcium in resting and stimulated renal epithelial LLC-PK1 cells. The cells were exposed for 5 minutes to a buffer containing 1.8 mM of the stable isotope 44 Ca in the presence of 1 µM of the calcium ionophore A23187 (middle row) and 100 nM of the arginine vasopressin hormone (bottom row).

    Techniques Used:

    SIMS images of 39 K, 23 Na, and 40 Ca revealing intracellular distributions of potassium, sodium, and calcium in renal epithelial LLC-PK1 cells. The cells were treated with 1 µM nocodazole for 4 hours and 100 nM taxol for 18 hours.
    Figure Legend Snippet: SIMS images of 39 K, 23 Na, and 40 Ca revealing intracellular distributions of potassium, sodium, and calcium in renal epithelial LLC-PK1 cells. The cells were treated with 1 µM nocodazole for 4 hours and 100 nM taxol for 18 hours.

    Techniques Used:

    SIMS images showing the distributions of 39 K, 23 Na, 40 Ca, and 12 C in fractured freeze-dried renal epithelial LLC-PK1 cells.
    Figure Legend Snippet: SIMS images showing the distributions of 39 K, 23 Na, 40 Ca, and 12 C in fractured freeze-dried renal epithelial LLC-PK1 cells.

    Techniques Used:

    18) Product Images from "Ciliotherapy: Remote Control of Primary Cilia Movement and Function by Magnetic Nanoparticles"

    Article Title: Ciliotherapy: Remote Control of Primary Cilia Movement and Function by Magnetic Nanoparticles

    Journal: ACS nano

    doi: 10.1021/acsnano.9b00033

    The CT-Fe 2 O 3 -NPs specifically target primary cilia under flow conditions and improve cilia structure and function. (A) A single-cell–single-cilium analysis was performed in a live cell to quantify the targeting specificity of the CT-Fe 2 O 3 -NPs at different time points (0 to 120 min). The top panel shows DIC images to confirm the presence of a cilium and fluorescence images to verify the CT-Fe 2 O 3 -NP specificity. The bottom panel shows the fluorescence images of the CT-Fe 2 O 3 -NPs alone. The CT-Fe 2 O 3 -NP fluorescence intensity per area ( I / μ m 2 ) was quantified in cilia vs the cell body and the cell body vs background fluorescence. A direct recording of the CT-Fe 2 O 3 . (B) Prussian blue staining confirmed the direct and specific binding of the CT-Fe 2 O 3 -NPs to the primary cilia. (C) Fluorescence images show that fenoldopam and the CT-Fe 2 O 3 -NPs (red) increased the cilia length (16 h of treatment) compared with controls (PBS treatment or cCT-Fe 2 O 3 -NPs). The ciliary marker acetylated- α . (D) Representative dot-plotted bar graph showing the ciliary lengths measured in cells receiving different treatments (acquired from 5 preparations in each group; a minimum of 10 cilia were randomly selected from each preparation). (E) An external magnetic field acting on the CT-Fe 2 O 3 -NPs (CT-M-Fe 2 O 3 ). (F) 5HT 6 -mCherry-GGECO1.0 was expressed in LLC-PK1 cells to measure cytosolic and intraciliary Ca 2+ signaling. GFP was used to measure changes in Ca 2+ signals, mCherry was used to normalize motion artifacts, and DIC was used to track cilia movement. The GFP/mCherry ratio (pseudocolored) indicates normalized Ca 2+ levels. Images of cells before and after challenge with either fluid flow (CT-Fe 2 O 3 -NPs) or the magnetic field (CT-M-Fe 2 O 3 -NPs) are shown ( N = 6, 30 fps). The colored bar shows the Ca 2+ . (G) An image of the cumulative intensity profile (achieved by ND acquisition, Nikon system) shows high cellular and ciliary Ca 2+ levels when cells were exposed to an external magnetic force. (H) Average cytosolic (red) and cilioplasmic (blue) Ca 2+ levels (in arbitrary units) are shown in line graphs. (I) Kymograph analysis of Ca 2+ signaling in the cell body and cilia was performed in cells treated with the control (cCT-M-Fe 2 O 3 -NPs) and the CT-M-Fe 2 O 3 -NPs. The colored bar shows the Ca 2+ levels. In all cases, vehicle (PBS) and superparamagnetic Fe 2 O 3 -NPs without loaded drug in the absence (cCT-Fe 2 O 3 -NPs) or presence (cCT-M-Fe 2 O 3- NPs) of the magnetic field were used as controls. N = 4 samples per group in each study. ****, p
    Figure Legend Snippet: The CT-Fe 2 O 3 -NPs specifically target primary cilia under flow conditions and improve cilia structure and function. (A) A single-cell–single-cilium analysis was performed in a live cell to quantify the targeting specificity of the CT-Fe 2 O 3 -NPs at different time points (0 to 120 min). The top panel shows DIC images to confirm the presence of a cilium and fluorescence images to verify the CT-Fe 2 O 3 -NP specificity. The bottom panel shows the fluorescence images of the CT-Fe 2 O 3 -NPs alone. The CT-Fe 2 O 3 -NP fluorescence intensity per area ( I / μ m 2 ) was quantified in cilia vs the cell body and the cell body vs background fluorescence. A direct recording of the CT-Fe 2 O 3 . (B) Prussian blue staining confirmed the direct and specific binding of the CT-Fe 2 O 3 -NPs to the primary cilia. (C) Fluorescence images show that fenoldopam and the CT-Fe 2 O 3 -NPs (red) increased the cilia length (16 h of treatment) compared with controls (PBS treatment or cCT-Fe 2 O 3 -NPs). The ciliary marker acetylated- α . (D) Representative dot-plotted bar graph showing the ciliary lengths measured in cells receiving different treatments (acquired from 5 preparations in each group; a minimum of 10 cilia were randomly selected from each preparation). (E) An external magnetic field acting on the CT-Fe 2 O 3 -NPs (CT-M-Fe 2 O 3 ). (F) 5HT 6 -mCherry-GGECO1.0 was expressed in LLC-PK1 cells to measure cytosolic and intraciliary Ca 2+ signaling. GFP was used to measure changes in Ca 2+ signals, mCherry was used to normalize motion artifacts, and DIC was used to track cilia movement. The GFP/mCherry ratio (pseudocolored) indicates normalized Ca 2+ levels. Images of cells before and after challenge with either fluid flow (CT-Fe 2 O 3 -NPs) or the magnetic field (CT-M-Fe 2 O 3 -NPs) are shown ( N = 6, 30 fps). The colored bar shows the Ca 2+ . (G) An image of the cumulative intensity profile (achieved by ND acquisition, Nikon system) shows high cellular and ciliary Ca 2+ levels when cells were exposed to an external magnetic force. (H) Average cytosolic (red) and cilioplasmic (blue) Ca 2+ levels (in arbitrary units) are shown in line graphs. (I) Kymograph analysis of Ca 2+ signaling in the cell body and cilia was performed in cells treated with the control (cCT-M-Fe 2 O 3 -NPs) and the CT-M-Fe 2 O 3 -NPs. The colored bar shows the Ca 2+ levels. In all cases, vehicle (PBS) and superparamagnetic Fe 2 O 3 -NPs without loaded drug in the absence (cCT-Fe 2 O 3 -NPs) or presence (cCT-M-Fe 2 O 3- NPs) of the magnetic field were used as controls. N = 4 samples per group in each study. ****, p

    Techniques Used: Fluorescence, Staining, Binding Assay, Marker

    19) Product Images from "Amphotericin B-Induced Renal Tubular Cell Injury Is Mediated by Na+ Influx through Ion-Permeable Pores and Subsequent Activation of Mitogen-Activated Protein Kinases and Elevation of Intracellular Ca2+ Concentration ▿"

    Article Title: Amphotericin B-Induced Renal Tubular Cell Injury Is Mediated by Na+ Influx through Ion-Permeable Pores and Subsequent Activation of Mitogen-Activated Protein Kinases and Elevation of Intracellular Ca2+ Concentration ▿

    Journal: Antimicrobial Agents and Chemotherapy

    doi: 10.1128/AAC.01137-08

    Effects of MAPK inhibitors on the AMB-induced loss of cell viability in LLC-PK1 cells. Cells were exposed to 15 μg/ml AMB for 6 h in Krebs-Ringer buffer. The p38 MAPK inhibitor PD169316 (10 μM), JNKI (10 μM), and the ERK inhibitor
    Figure Legend Snippet: Effects of MAPK inhibitors on the AMB-induced loss of cell viability in LLC-PK1 cells. Cells were exposed to 15 μg/ml AMB for 6 h in Krebs-Ringer buffer. The p38 MAPK inhibitor PD169316 (10 μM), JNKI (10 μM), and the ERK inhibitor

    Techniques Used:

    Effects of Ca 2+ modulators of ER (A) and mitochondria (B) on AMB-induced loss of cell viability in LLC-PK1 cells. Cells were exposed to AMB (15 μg/ml) for 6 h in Krebs-Ringer buffer, and their viability was assessed by the WST-8 assay.
    Figure Legend Snippet: Effects of Ca 2+ modulators of ER (A) and mitochondria (B) on AMB-induced loss of cell viability in LLC-PK1 cells. Cells were exposed to AMB (15 μg/ml) for 6 h in Krebs-Ringer buffer, and their viability was assessed by the WST-8 assay.

    Techniques Used:

    Concentration-dependent and time-dependent decreases in tissue ATP content after exposure of LLC-PK1 cells to AMB. Cells were exposed to various concentrations of AMB for 24 h. The data represent the means ± SEMs of four to eight experiments.
    Figure Legend Snippet: Concentration-dependent and time-dependent decreases in tissue ATP content after exposure of LLC-PK1 cells to AMB. Cells were exposed to various concentrations of AMB for 24 h. The data represent the means ± SEMs of four to eight experiments.

    Techniques Used: Concentration Assay

    Concentration-dependent and time-dependent cell injury to LLC-PK1 cells induced by AMB, as assessed by the WST-8 assay or determination of the level of LDH leakage (A) and staining with the representative stains propidium iodide (PI) and TUNEL (B). Cells
    Figure Legend Snippet: Concentration-dependent and time-dependent cell injury to LLC-PK1 cells induced by AMB, as assessed by the WST-8 assay or determination of the level of LDH leakage (A) and staining with the representative stains propidium iodide (PI) and TUNEL (B). Cells

    Techniques Used: Concentration Assay, Staining, TUNEL Assay

    Inhibition by Mβ-CD of AMB-induced loss of cell viability in LLC-PK1 cells and its reversal of the inhibition by cholesterol. Cholesterol depletion was carried out by incubating the cells with Mβ-CD for 1 h at 37°C. Cholesterol
    Figure Legend Snippet: Inhibition by Mβ-CD of AMB-induced loss of cell viability in LLC-PK1 cells and its reversal of the inhibition by cholesterol. Cholesterol depletion was carried out by incubating the cells with Mβ-CD for 1 h at 37°C. Cholesterol

    Techniques Used: Inhibition

    Inhibition of the AMB-induced loss of cell viability in LLC-PK1 cells by lowering the extracellular Na + concentration (A) or the chelation of intracellular Ca 2+ with BAPTA-AM (B). Cells were exposed to AMB (15 μg/ml) for 6 h in
    Figure Legend Snippet: Inhibition of the AMB-induced loss of cell viability in LLC-PK1 cells by lowering the extracellular Na + concentration (A) or the chelation of intracellular Ca 2+ with BAPTA-AM (B). Cells were exposed to AMB (15 μg/ml) for 6 h in

    Techniques Used: Inhibition, Concentration Assay

    Time courses of phospho-JNK, phospho-p38, and phospho-ERK activation induced by AMB in LLC-PK1 cells. Cells were stimulated with AMB (15 μg/ml) for the indicated times. Phospho-JNK, phospho-p38, and phospho-ERK activation was determined by Western
    Figure Legend Snippet: Time courses of phospho-JNK, phospho-p38, and phospho-ERK activation induced by AMB in LLC-PK1 cells. Cells were stimulated with AMB (15 μg/ml) for the indicated times. Phospho-JNK, phospho-p38, and phospho-ERK activation was determined by Western

    Techniques Used: Activation Assay, Western Blot

    20) Product Images from "myo-Inositol Oxygenase Overexpression Accentuates Generation of Reactive Oxygen Species and Exacerbates Cellular Injury following High Glucose Ambience"

    Article Title: myo-Inositol Oxygenase Overexpression Accentuates Generation of Reactive Oxygen Species and Exacerbates Cellular Injury following High Glucose Ambience

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M115.669952

    A and B , effect of MIOX overexpression on TGF-β1 expression and bioactivity under HG ambience in LLC-PK1 cells. No significant increase in the expression or bioactivity was observed with transfection of EV (pcDNA) under basal LG conditions ( columns
    Figure Legend Snippet: A and B , effect of MIOX overexpression on TGF-β1 expression and bioactivity under HG ambience in LLC-PK1 cells. No significant increase in the expression or bioactivity was observed with transfection of EV (pcDNA) under basal LG conditions ( columns

    Techniques Used: Over Expression, Expressing, Transfection

    Expression and activity of MIOX in LLC-PK1 cells following its overexpression in HG ambience. Western blotting analyses show a dose-dependent increased expression of MIOX in response to various d -glucose concentrations, whereas treatment with MIOX-specific
    Figure Legend Snippet: Expression and activity of MIOX in LLC-PK1 cells following its overexpression in HG ambience. Western blotting analyses show a dose-dependent increased expression of MIOX in response to various d -glucose concentrations, whereas treatment with MIOX-specific

    Techniques Used: Expressing, Activity Assay, Over Expression, Western Blot

    21) Product Images from "Meclizine Preconditioning Protects the Kidney Against Ischemia–Reperfusion Injury"

    Article Title: Meclizine Preconditioning Protects the Kidney Against Ischemia–Reperfusion Injury

    Journal: EBioMedicine

    doi: 10.1016/j.ebiom.2015.07.035

    Meclizine up-regulates glycolysis in glucose containing media and reduces cellular ATP levels in galactose media. (A) LDH release, (B) Cell viability and (C) cellular ATP levels of LLC-PK1 cells cultured under 21% or 5% O 2 with 25 mM glucose or 10 mM galactose for 24 h (n = 3). (D) Lactate production in HK-2 cells after treatment with or without 25 μM of meclizine for 17 h in 21% O 2 and glucose containing media (n = 4). (E) Real-time PCR analysis of Slca2 , Pgk1 , Hk2 and Ldha mRNA after treatment with or without 25 μM of meclizine for 17 h (n = 3). (F) Cellular ATP levels in HK-2 cells treated with or without 25 μM of meclizine for 17 h and then cultured with 10 mM galactose (n = 3). (G) Western blot displays protein level of HIF1-α after meclizine or CoCl 2 pretreatment. (H) Quantitation of the band density of HIF1-α (n = 3). **p
    Figure Legend Snippet: Meclizine up-regulates glycolysis in glucose containing media and reduces cellular ATP levels in galactose media. (A) LDH release, (B) Cell viability and (C) cellular ATP levels of LLC-PK1 cells cultured under 21% or 5% O 2 with 25 mM glucose or 10 mM galactose for 24 h (n = 3). (D) Lactate production in HK-2 cells after treatment with or without 25 μM of meclizine for 17 h in 21% O 2 and glucose containing media (n = 4). (E) Real-time PCR analysis of Slca2 , Pgk1 , Hk2 and Ldha mRNA after treatment with or without 25 μM of meclizine for 17 h (n = 3). (F) Cellular ATP levels in HK-2 cells treated with or without 25 μM of meclizine for 17 h and then cultured with 10 mM galactose (n = 3). (G) Western blot displays protein level of HIF1-α after meclizine or CoCl 2 pretreatment. (H) Quantitation of the band density of HIF1-α (n = 3). **p

    Techniques Used: Cell Culture, Real-time Polymerase Chain Reaction, Western Blot, Quantitation Assay

    Cellular phosphoethanolamine is increased by meclizine and recapitulated meclizine-induced protection. (A) Phosphoethanolamine levels in HK-2 cells treated with or without 25 μM of meclizine for 17 h (n = 3). (B) Cellular ATP levels in HK-2 cells cultured in 10% DMEM containing 10 mM galactose with or without 10 μM of ethanolamine (EA) for 17 h (n = 4). (C) Lactate production in HK-2 cells after treatment with or without 10 μM of EA for 17 h (n = 7). (D) LDH release from HK-2 cells treated with either 10 or 100 μM of EA for 17 h followed by 2 hr of chemical anoxia (n = 7). (E) LDH release from LLC-PK1 cells treated with 10 μM of EA for 17 h followed by 2 hr of chemical anoxia (n = 4). (F) BUN and (G) serum creatinine concentrations at 24 and 48 h after IRI treated 2 h before, just after clamp removal and skin closure 24 h after ischemia with vehicle (n = 5) or EA (n = 4). (H) Representative images of H E and PAS-stained kidney sections 48 h after IRI. Original magnification 100 ×, scale bar = 100 μm. (I) Summary of the mechanisms proposed for meclizine-induced protective effects against ischemic injury. Meclizine inhibits phosphate cytidylyltransferase 2 (PCYT2) and causes an increase in cytosolic phosphoethanolamine, a central precursor in the Kennedy pathway. High levels of intracellular phosphoethanolamine inhibit mitochondrial respiration. **p
    Figure Legend Snippet: Cellular phosphoethanolamine is increased by meclizine and recapitulated meclizine-induced protection. (A) Phosphoethanolamine levels in HK-2 cells treated with or without 25 μM of meclizine for 17 h (n = 3). (B) Cellular ATP levels in HK-2 cells cultured in 10% DMEM containing 10 mM galactose with or without 10 μM of ethanolamine (EA) for 17 h (n = 4). (C) Lactate production in HK-2 cells after treatment with or without 10 μM of EA for 17 h (n = 7). (D) LDH release from HK-2 cells treated with either 10 or 100 μM of EA for 17 h followed by 2 hr of chemical anoxia (n = 7). (E) LDH release from LLC-PK1 cells treated with 10 μM of EA for 17 h followed by 2 hr of chemical anoxia (n = 4). (F) BUN and (G) serum creatinine concentrations at 24 and 48 h after IRI treated 2 h before, just after clamp removal and skin closure 24 h after ischemia with vehicle (n = 5) or EA (n = 4). (H) Representative images of H E and PAS-stained kidney sections 48 h after IRI. Original magnification 100 ×, scale bar = 100 μm. (I) Summary of the mechanisms proposed for meclizine-induced protective effects against ischemic injury. Meclizine inhibits phosphate cytidylyltransferase 2 (PCYT2) and causes an increase in cytosolic phosphoethanolamine, a central precursor in the Kennedy pathway. High levels of intracellular phosphoethanolamine inhibit mitochondrial respiration. **p

    Techniques Used: Cell Culture, Staining

    Meclizine attenuates LDH and cytochrome c release during 2-DG and NaCN treatment of tubular epithelial cells. (A) LDH release from LLC-PK1 cells treated with different concentrations of meclizine for 17 h followed by 2hr of chemical anoxia (n = 3). (B) LDH release from HK-2 cells treated with different concentrations of meclizine for 17 h followed by 2hr of chemical anoxia (n = 7). (C) Western blot shows released cytochrome c from HK-2 cells after chemical anoxia in the presence or absence of meclizine pretreatment. (D) Quantitation of the band density of cytochrome c (n = 3). *p
    Figure Legend Snippet: Meclizine attenuates LDH and cytochrome c release during 2-DG and NaCN treatment of tubular epithelial cells. (A) LDH release from LLC-PK1 cells treated with different concentrations of meclizine for 17 h followed by 2hr of chemical anoxia (n = 3). (B) LDH release from HK-2 cells treated with different concentrations of meclizine for 17 h followed by 2hr of chemical anoxia (n = 7). (C) Western blot shows released cytochrome c from HK-2 cells after chemical anoxia in the presence or absence of meclizine pretreatment. (D) Quantitation of the band density of cytochrome c (n = 3). *p

    Techniques Used: Western Blot, Quantitation Assay

    22) Product Images from "Protective Effect of Standardized Extract of Ginkgo biloba against Cisplatin-Induced Nephrotoxicity"

    Article Title: Protective Effect of Standardized Extract of Ginkgo biloba against Cisplatin-Induced Nephrotoxicity

    Journal: Evidence-based Complementary and Alternative Medicine : eCAM

    doi: 10.1155/2013/846126

    EGb repressed CDDP-stimulated generation of ROS in LLC-PK1 cells. (a) LLC-PK1 cells were incubated with 50 μ M CDDP for the indicated time periods. (b) LLC-PK1 cells were preincubated with EGb (200, 100, and 50 μ g/mL) for 24 h and then incubated with 50 μ M CDDP at 37°C for 12 h. The generation of ROS was measured by using the fluorescent dye DCF-DA in FACScan flow cytometry. The corresponding linear diagram of FACScan flow cytometry is shown. Data represent mean ± S.D. from three independent experiments. * P
    Figure Legend Snippet: EGb repressed CDDP-stimulated generation of ROS in LLC-PK1 cells. (a) LLC-PK1 cells were incubated with 50 μ M CDDP for the indicated time periods. (b) LLC-PK1 cells were preincubated with EGb (200, 100, and 50 μ g/mL) for 24 h and then incubated with 50 μ M CDDP at 37°C for 12 h. The generation of ROS was measured by using the fluorescent dye DCF-DA in FACScan flow cytometry. The corresponding linear diagram of FACScan flow cytometry is shown. Data represent mean ± S.D. from three independent experiments. * P

    Techniques Used: Incubation, Flow Cytometry

    EGb suppressed CDDP-induced I κ B degradation, p65 phosphorylation, and iNOS expression in LLC-PK1 cells. (a) LLC-PK1 cells were preincubated with EGb (200, 100, and 50 μ g/mL) for 24 h and then incubated with 50 μ M CDDP at 37°C for 12 h. I κ B, iNOS, phospho-p65, p65, caspase-3, caspase-9, and caspase-8 protein levels from LLC-PK1 cells extracts were analyzed by western blotting. (b) Protein expressions were semiquantified by densitometry analysis. (c) iNOS mRNA levels were analyzed with RT-PCR and then semiquantified. Data represent mean ± S.D. from three independent experiments. * P
    Figure Legend Snippet: EGb suppressed CDDP-induced I κ B degradation, p65 phosphorylation, and iNOS expression in LLC-PK1 cells. (a) LLC-PK1 cells were preincubated with EGb (200, 100, and 50 μ g/mL) for 24 h and then incubated with 50 μ M CDDP at 37°C for 12 h. I κ B, iNOS, phospho-p65, p65, caspase-3, caspase-9, and caspase-8 protein levels from LLC-PK1 cells extracts were analyzed by western blotting. (b) Protein expressions were semiquantified by densitometry analysis. (c) iNOS mRNA levels were analyzed with RT-PCR and then semiquantified. Data represent mean ± S.D. from three independent experiments. * P

    Techniques Used: Expressing, Incubation, Western Blot, Reverse Transcription Polymerase Chain Reaction

    23) Product Images from "Nigratine as first-in-class dual inhibitor of necroptosis and ferroptosis regulated cell death"

    Article Title: Nigratine as first-in-class dual inhibitor of necroptosis and ferroptosis regulated cell death

    Journal: bioRxiv

    doi: 10.1101/2020.12.15.422885

    Nigratine protects porcine kidney epithelial LLC-PK1 cell line from lipid peroxidation and cell death triggered by RSL3. LLC-PK1 cell line was treated with 2µM of RSL3 and increasing concentration of Nigratine or ferrostatin-1 (Fer-1). ( a ) Cell viability was estimated by MTS assay. Data are shown as the mean ± SD of three replicates. ( b ) Lipid peroxidation was detected by cellular BODIPY 581/591 C11 staining. Fluorescence was recorded with the IncuCyte S3 live cell imaging apparatus. Data are shown as the mean ± SD of three replicates of nine replicates. ( c ) Representative phase-contrast and fluorescence images of cells stained with BODIPY 581/591 C11 probe are visualized using the IncuCyte S3 live cell imaging apparatus
    Figure Legend Snippet: Nigratine protects porcine kidney epithelial LLC-PK1 cell line from lipid peroxidation and cell death triggered by RSL3. LLC-PK1 cell line was treated with 2µM of RSL3 and increasing concentration of Nigratine or ferrostatin-1 (Fer-1). ( a ) Cell viability was estimated by MTS assay. Data are shown as the mean ± SD of three replicates. ( b ) Lipid peroxidation was detected by cellular BODIPY 581/591 C11 staining. Fluorescence was recorded with the IncuCyte S3 live cell imaging apparatus. Data are shown as the mean ± SD of three replicates of nine replicates. ( c ) Representative phase-contrast and fluorescence images of cells stained with BODIPY 581/591 C11 probe are visualized using the IncuCyte S3 live cell imaging apparatus

    Techniques Used: Concentration Assay, MTS Assay, Staining, Fluorescence, Live Cell Imaging

    24) Product Images from "Coronavirus Endoribonuclease Activity in Porcine Epidemic Diarrhea Virus Suppresses Type I and Type III Interferon Responses"

    Article Title: Coronavirus Endoribonuclease Activity in Porcine Epidemic Diarrhea Virus Suppresses Type I and Type III Interferon Responses

    Journal: Journal of Virology

    doi: 10.1128/JVI.02000-18

    EndoU-deficient PEDV induces early and robust type I and III IFN responses in PK1 cells. (A and B) PK1 cells were transfected with 200 ng poly(I:C) for 24 h. Total RNA was extracted and the relative mRNA levels (compared to levels in mock-treated cells) of (A) IFN-β and IFN-λ3 and (B) ISGs were measured by reverse transcription-quantitative PCR (RT-qPCR). (C to E) PK1 cells were infected with either icPEDV-wt or icPEDV-EnUmt (0.1 TCID 50 /cell). The relative mRNA levels (comparing to levels in WT-infected cells at 8 h postinfection) of IFN-β (C) and IFN-λ3 (D) were presented as fold changes. The level of N gene (E) relative to GAPDH mRNA were expressed as 2 −ΔCT [Δ C T = C T(gene of interest) −C T(β-actin) ]. (F) PK1 cells were infected with PEDV (0.1 TCID 50 /cell) and culture supernatant was collected at indicated time points and titrated on Vero cells. The experiment was performed three times and the representative data are shown. Error bars represent mean ± standard deviation (SD). *, P
    Figure Legend Snippet: EndoU-deficient PEDV induces early and robust type I and III IFN responses in PK1 cells. (A and B) PK1 cells were transfected with 200 ng poly(I:C) for 24 h. Total RNA was extracted and the relative mRNA levels (compared to levels in mock-treated cells) of (A) IFN-β and IFN-λ3 and (B) ISGs were measured by reverse transcription-quantitative PCR (RT-qPCR). (C to E) PK1 cells were infected with either icPEDV-wt or icPEDV-EnUmt (0.1 TCID 50 /cell). The relative mRNA levels (comparing to levels in WT-infected cells at 8 h postinfection) of IFN-β (C) and IFN-λ3 (D) were presented as fold changes. The level of N gene (E) relative to GAPDH mRNA were expressed as 2 −ΔCT [Δ C T = C T(gene of interest) −C T(β-actin) ]. (F) PK1 cells were infected with PEDV (0.1 TCID 50 /cell) and culture supernatant was collected at indicated time points and titrated on Vero cells. The experiment was performed three times and the representative data are shown. Error bars represent mean ± standard deviation (SD). *, P

    Techniques Used: Transfection, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Infection, Standard Deviation

    EndoU-deficient PEDV infection induces robust expression of ISGs and proinflammatory cytokines in PK1 cells. PK1 cells were infected with either icPEDV-wt or icPEDV-EnUmt (0.1 TCID 50 /cell), and cells were harvested for total RNA extraction at indicated time points. The relative mRNA levels (comparing to those in mock-infected cells at 12 h postinfection) of ISG54 (A), ISG15 (B), 2′-5′-oligoadenylate synthetase 1 (OAS1) (C), and tumor necrosis factor alpha (TNF-α) (D) were measured using RT-qPCR. The experiment was performed three times, and the representative data are shown. Error bars represent mean ± SD. ****, P
    Figure Legend Snippet: EndoU-deficient PEDV infection induces robust expression of ISGs and proinflammatory cytokines in PK1 cells. PK1 cells were infected with either icPEDV-wt or icPEDV-EnUmt (0.1 TCID 50 /cell), and cells were harvested for total RNA extraction at indicated time points. The relative mRNA levels (comparing to those in mock-infected cells at 12 h postinfection) of ISG54 (A), ISG15 (B), 2′-5′-oligoadenylate synthetase 1 (OAS1) (C), and tumor necrosis factor alpha (TNF-α) (D) were measured using RT-qPCR. The experiment was performed three times, and the representative data are shown. Error bars represent mean ± SD. ****, P

    Techniques Used: Infection, Expressing, RNA Extraction, Quantitative RT-PCR

    25) Product Images from "A snake toxin as a theranostic agent for the type 2 vasopressin receptor"

    Article Title: A snake toxin as a theranostic agent for the type 2 vasopressin receptor

    Journal: Theranostics

    doi: 10.7150/thno.47485

    Labeling of endogenously expressed V2R in the LLC-PK1 cell line, in either fresh or PFA-fixed cells by (A) 100 nM of AFD-488-MQ1, (B) 100 nM of Cy5.5-MQ1. Specificity is determined by the last panels in the presence of either 3.4 µM MQ1 or 1 µM SR121463. (C) Cy5.5-MQ1 labeling at 100 nM in wild-type CHO cells (CHO WT), CHO cells stably expressing human V2R (CHO hV2R) and in renal cancer cell lines CAKI2, ACHN and A498 expressing hV2R. (D) Specificity is determined in the presence of 3.4 µM MQ1.
    Figure Legend Snippet: Labeling of endogenously expressed V2R in the LLC-PK1 cell line, in either fresh or PFA-fixed cells by (A) 100 nM of AFD-488-MQ1, (B) 100 nM of Cy5.5-MQ1. Specificity is determined by the last panels in the presence of either 3.4 µM MQ1 or 1 µM SR121463. (C) Cy5.5-MQ1 labeling at 100 nM in wild-type CHO cells (CHO WT), CHO cells stably expressing human V2R (CHO hV2R) and in renal cancer cell lines CAKI2, ACHN and A498 expressing hV2R. (D) Specificity is determined in the presence of 3.4 µM MQ1.

    Techniques Used: Labeling, Stable Transfection, Expressing

    26) Product Images from "Chimeric Porcine Deltacoronaviruses with Sparrow Coronavirus Spike Protein or the Receptor-Binding Domain Infect Pigs but Lose Virulence and Intestinal Tropism"

    Article Title: Chimeric Porcine Deltacoronaviruses with Sparrow Coronavirus Spike Protein or the Receptor-Binding Domain Infect Pigs but Lose Virulence and Intestinal Tropism

    Journal: Viruses

    doi: 10.3390/v13010122

    Characterization of the recombinant PDCoVs. ( A ) Multistep growth kinetics of the infectious clone-derived recombinant PDCoVs (icPDCoV, icPDCoV-RBD ISU , icPDCoV-S HKU17 ), and the parental PDCoV OH-FD22 strain. LLC-PK1 cells were inoculated with each virus at a MOI of 0.01. Supernatants were sampled at different time points and titrated for TCID 50. ( B ) The infectivity of the recombinant viruses in wildtype ST (ST-WT) cells and APN-knockout ST (ST-APN-KO) cells. TGEV and PEDV were used as APN-dependent and APN-independent controls, respectively. Monolayer of cells infected with different viruses with MOI = 0.2. After removal of the inoculum, the cells were cultured for additional 8 h, fixed and stained for TGEV, PEDV, and PDCoV, respectively. Fluorescent focus units (FFU) were counted using a fluorescent microscope. Data are presented as the means ± SD. *** p
    Figure Legend Snippet: Characterization of the recombinant PDCoVs. ( A ) Multistep growth kinetics of the infectious clone-derived recombinant PDCoVs (icPDCoV, icPDCoV-RBD ISU , icPDCoV-S HKU17 ), and the parental PDCoV OH-FD22 strain. LLC-PK1 cells were inoculated with each virus at a MOI of 0.01. Supernatants were sampled at different time points and titrated for TCID 50. ( B ) The infectivity of the recombinant viruses in wildtype ST (ST-WT) cells and APN-knockout ST (ST-APN-KO) cells. TGEV and PEDV were used as APN-dependent and APN-independent controls, respectively. Monolayer of cells infected with different viruses with MOI = 0.2. After removal of the inoculum, the cells were cultured for additional 8 h, fixed and stained for TGEV, PEDV, and PDCoV, respectively. Fluorescent focus units (FFU) were counted using a fluorescent microscope. Data are presented as the means ± SD. *** p

    Techniques Used: Recombinant, Derivative Assay, Infection, Knock-Out, Cell Culture, Staining, Microscopy

    The rescue of the recombinant PDCoVs (magnification, 100×). LLC-PK1 cells transfected with N transcript only ( A ) and the full-length mRNA genome and N transcript of PDCoV ( B ) at 72 h post electroporation (hpi) viewed by light microscopy. IF staining for cells inoculated with control ( C ), icPDCoV ( D ), icPDCoV-RBD ISU ( E ), and icPDCoV-S HKU17 ( F ) at 24 hpi [PDCoV N gene (in green) and nuclei (in blue)].
    Figure Legend Snippet: The rescue of the recombinant PDCoVs (magnification, 100×). LLC-PK1 cells transfected with N transcript only ( A ) and the full-length mRNA genome and N transcript of PDCoV ( B ) at 72 h post electroporation (hpi) viewed by light microscopy. IF staining for cells inoculated with control ( C ), icPDCoV ( D ), icPDCoV-RBD ISU ( E ), and icPDCoV-S HKU17 ( F ) at 24 hpi [PDCoV N gene (in green) and nuclei (in blue)].

    Techniques Used: Recombinant, Transfection, Electroporation, Light Microscopy, Staining

    27) Product Images from "High Basolateral Glucose Increases Sodium-Glucose Cotransporter 2 and Reduces Sirtuin-1 in Renal Tubules through Glucose Transporter-2 Detection"

    Article Title: High Basolateral Glucose Increases Sodium-Glucose Cotransporter 2 and Reduces Sirtuin-1 in Renal Tubules through Glucose Transporter-2 Detection

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-25054-y

    The GLUT2/importin-α1/HNF-1α pathway mediates high-glucose-induced SGLT2 expression. ( A ) Quantitative real-time PCR analysis of the relative abundance of mRNAs encoding importins in LLC-PK1 cells. Real-time PCR data were normalized to those of the GAPDH mRNA. Relative fold-differences were calculated using the mean value ( n = 6) of importin-α6. ( B ) Immunoblotting confirmed the efficiency of siRNA knockdown of each of the representative importin-α isoform that was abundantly expressed in PTs. Results are representative of four independent experiments. ( C ) Subcellular fractionation and immunoblotting analysis of the localization of HNF-1α. LLC-PK1 cells were transfected with the indicated siRNA duplexes and 48 h later, cells were treated with media containing high glucose levels. Cytoplasmic and nuclear lysates were collected and analyzed using immunoblotting with an anti-HNF-1α antibody. Results are representative of four independent experiments. ( D ) LLC-PK1 cells were transiently transfected with siRNAs targeting importin-α1 or a non-targeting control siRNA. Whole cell lysates were prepared 48 h post-transfection and analyzed using immunoblotting. Results are representative of four independent experiments. The bar graph represents the band intensity of each group (* P
    Figure Legend Snippet: The GLUT2/importin-α1/HNF-1α pathway mediates high-glucose-induced SGLT2 expression. ( A ) Quantitative real-time PCR analysis of the relative abundance of mRNAs encoding importins in LLC-PK1 cells. Real-time PCR data were normalized to those of the GAPDH mRNA. Relative fold-differences were calculated using the mean value ( n = 6) of importin-α6. ( B ) Immunoblotting confirmed the efficiency of siRNA knockdown of each of the representative importin-α isoform that was abundantly expressed in PTs. Results are representative of four independent experiments. ( C ) Subcellular fractionation and immunoblotting analysis of the localization of HNF-1α. LLC-PK1 cells were transfected with the indicated siRNA duplexes and 48 h later, cells were treated with media containing high glucose levels. Cytoplasmic and nuclear lysates were collected and analyzed using immunoblotting with an anti-HNF-1α antibody. Results are representative of four independent experiments. ( D ) LLC-PK1 cells were transiently transfected with siRNAs targeting importin-α1 or a non-targeting control siRNA. Whole cell lysates were prepared 48 h post-transfection and analyzed using immunoblotting. Results are representative of four independent experiments. The bar graph represents the band intensity of each group (* P

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction, Fractionation, Transfection

    Effect of Cana on the expression of SIRT1 and its downstream signaling components. Confluent growth-arrested LLC-PK1 cell monolayers were stimulated with HG medium on the basolateral side for up to 24 h with or without pretreatment with Cana in the apical side. Immunofluorescence analysis ( A ) and immunoblotting ( B ) for SGLT2 expression in LLC-PK1 cells. The relative quantification of the SGLT2 immunofluorescence was measured and is indicated in the bar graphs. Scale bar, 50 µm. * P
    Figure Legend Snippet: Effect of Cana on the expression of SIRT1 and its downstream signaling components. Confluent growth-arrested LLC-PK1 cell monolayers were stimulated with HG medium on the basolateral side for up to 24 h with or without pretreatment with Cana in the apical side. Immunofluorescence analysis ( A ) and immunoblotting ( B ) for SGLT2 expression in LLC-PK1 cells. The relative quantification of the SGLT2 immunofluorescence was measured and is indicated in the bar graphs. Scale bar, 50 µm. * P

    Techniques Used: Expressing, Immunofluorescence

    28) Product Images from "Amphiphilic Cell-Penetrating Peptides Containing Natural and Unnatural Amino Acids as Drug Delivery Agents"

    Article Title: Amphiphilic Cell-Penetrating Peptides Containing Natural and Unnatural Amino Acids as Drug Delivery Agents

    Journal: Cells

    doi: 10.3390/cells11071156

    Cytotoxicity study of [DipR] 5 in MDA-MB-468, LLC-PK1, and MES- SA cells after 24 h ( A ) and 72 h; ( B ) incubation. Results are mean ± SD (n = 3) (NS = not significant, *** p
    Figure Legend Snippet: Cytotoxicity study of [DipR] 5 in MDA-MB-468, LLC-PK1, and MES- SA cells after 24 h ( A ) and 72 h; ( B ) incubation. Results are mean ± SD (n = 3) (NS = not significant, *** p

    Techniques Used: Multiple Displacement Amplification, Incubation

    29) Product Images from "Polycystin-2 (TRPP2) Regulates Primary Cilium Length in LLC-PK1 Renal Epithelial Cells"

    Article Title: Polycystin-2 (TRPP2) Regulates Primary Cilium Length in LLC-PK1 Renal Epithelial Cells

    Journal: bioRxiv

    doi: 10.1101/2020.02.24.962860

    Differences in the length of the primary cilium after inhibition of PKD2 gene expression. Bar graphs represent either the percent increase (positive bars) or decrease (negative bars) of the length of the primary cilium of LLC-PK1 cells in normal (1.2 mM) and high (6.2 mM) external Ca 2+ concentrations. Data were made relative to the Irss 1.2 mM Ca 2+ , control condition with the scrambled silencing RNA probe (Irss). Irss-treated cells responded to high external Ca 2+ with a large decrease in the length of the primary cilium. Ciliary length of PKD2 -silenced cells increased with respect to the it respective controls in the presence of both normal (1.2 mM) and high (6.2 mM) external Ca 2+ . Asterisks (***) indicate statistical significance at p
    Figure Legend Snippet: Differences in the length of the primary cilium after inhibition of PKD2 gene expression. Bar graphs represent either the percent increase (positive bars) or decrease (negative bars) of the length of the primary cilium of LLC-PK1 cells in normal (1.2 mM) and high (6.2 mM) external Ca 2+ concentrations. Data were made relative to the Irss 1.2 mM Ca 2+ , control condition with the scrambled silencing RNA probe (Irss). Irss-treated cells responded to high external Ca 2+ with a large decrease in the length of the primary cilium. Ciliary length of PKD2 -silenced cells increased with respect to the it respective controls in the presence of both normal (1.2 mM) and high (6.2 mM) external Ca 2+ . Asterisks (***) indicate statistical significance at p

    Techniques Used: Inhibition, Expressing

    Proposed model of primary cilium length regulation by PC2. Cartoon of the possible role(s) PC2 plays in the regulation of the length of the primary cilium in LLC-PK1 renal epithelial cells. Under physiological external Ca 2+ conditions (1.2 mM), the length primary cilium length is maintained by PC2-mediated Ca 2+ entry. In the presence of a high external Ca 2+ (6.2 mM) concentration, the increased intraciliary ion would help depolymerize axonemal microtubules, leading to reduction in their length, and thus that of the primary cilium. Either PC2 inhibition (by Li + or amiloride) or reduction of its expression (siRNA), would instead contribute to reducing Ca 2+ entry to the primary cilium, also reducing the rate MT depolymerization, rendering longer primary cilia.
    Figure Legend Snippet: Proposed model of primary cilium length regulation by PC2. Cartoon of the possible role(s) PC2 plays in the regulation of the length of the primary cilium in LLC-PK1 renal epithelial cells. Under physiological external Ca 2+ conditions (1.2 mM), the length primary cilium length is maintained by PC2-mediated Ca 2+ entry. In the presence of a high external Ca 2+ (6.2 mM) concentration, the increased intraciliary ion would help depolymerize axonemal microtubules, leading to reduction in their length, and thus that of the primary cilium. Either PC2 inhibition (by Li + or amiloride) or reduction of its expression (siRNA), would instead contribute to reducing Ca 2+ entry to the primary cilium, also reducing the rate MT depolymerization, rendering longer primary cilia.

    Techniques Used: Concentration Assay, Inhibition, Expressing

    Effect of PKD2 gene silencing on the length of the primary cilium of wild type LLC-PK1 cells. a. Primary cilia are observed in green (FITC) and nuclei stained in blue (DAPI) in cells transfected with scrambled (Irss) and PKD2-specific (P1ss) probes in either normal (1.2 mM) or high Ca 2+ (6.2 mM) conditions. In normal Ca 2+ , P1ss silenced cells had longer primary cilia than their respective controls (Irss, 1.2 Ca). Longer primary cilia were also observed in silenced P1ss cells in high Ca 2+ respect to Irss, 6.2 Ca condition. b. Box-Cox transformation for data of the length of the primary cilium in LLC-PK1 cells after inhibition of PKD2 gene expression under 1.2 mM (Top) and 6.2 mM (Bottom) external Ca 2+ conditions. Frequency data distributions are shown before (Left) and after (Right) Box-Cox transformation.
    Figure Legend Snippet: Effect of PKD2 gene silencing on the length of the primary cilium of wild type LLC-PK1 cells. a. Primary cilia are observed in green (FITC) and nuclei stained in blue (DAPI) in cells transfected with scrambled (Irss) and PKD2-specific (P1ss) probes in either normal (1.2 mM) or high Ca 2+ (6.2 mM) conditions. In normal Ca 2+ , P1ss silenced cells had longer primary cilia than their respective controls (Irss, 1.2 Ca). Longer primary cilia were also observed in silenced P1ss cells in high Ca 2+ respect to Irss, 6.2 Ca condition. b. Box-Cox transformation for data of the length of the primary cilium in LLC-PK1 cells after inhibition of PKD2 gene expression under 1.2 mM (Top) and 6.2 mM (Bottom) external Ca 2+ conditions. Frequency data distributions are shown before (Left) and after (Right) Box-Cox transformation.

    Techniques Used: Staining, Transfection, Transformation Assay, Inhibition, Expressing

    Effect of extracellular Ca 2+ on the length of the primary cilium of wild type LLC-PK1 cells. a. Acetylated-α-tubulin immunolabeling of confluent of LLC-PK1 cell monolayers was used to observe the primary cilium at normal (1.2 Ca, Top panel),and high (6.2 Ca) external Ca 2+ concentrations. Merged images also show cell nuclei by DAPI labeling (Blue). b. Histograms of ciliary length measurements in normal (1.2 mM, Top) and high (6.2 mM, Bottom) Ca 2+ concentration are shown before (Left), and after (Right), Box-Cox transformation. A not Normal left-skewed distribution of data is observed before transformation. Please note that Box-Cox transformed histograms require re-transforming the values to render primary cilia length in μm (see Materials Methods).
    Figure Legend Snippet: Effect of extracellular Ca 2+ on the length of the primary cilium of wild type LLC-PK1 cells. a. Acetylated-α-tubulin immunolabeling of confluent of LLC-PK1 cell monolayers was used to observe the primary cilium at normal (1.2 Ca, Top panel),and high (6.2 Ca) external Ca 2+ concentrations. Merged images also show cell nuclei by DAPI labeling (Blue). b. Histograms of ciliary length measurements in normal (1.2 mM, Top) and high (6.2 mM, Bottom) Ca 2+ concentration are shown before (Left), and after (Right), Box-Cox transformation. A not Normal left-skewed distribution of data is observed before transformation. Please note that Box-Cox transformed histograms require re-transforming the values to render primary cilia length in μm (see Materials Methods).

    Techniques Used: Immunolabeling, Labeling, Concentration Assay, Transformation Assay

    Primary cilia inmunolabeling and measurement. a. Confluent monolayers of wild-type LLC-PK1 cells were labeled with an anti-acetylated α-tubulin antibody (Green, Left Panel), and DAPI (Blue, Middle Panel). The merged image is shown on the Right. b. Primary cilia were identified (x40) and measured with the IPLab software. c. Top. Data distribution (Frequency) histograms before (Left panel) and after the Box-Cox transformation for the variable “Length of the primary cilium” are shown before and after Box-Cox transformation. The symmetry reached after the transformation is consistent with a Normalized distribution. Bottom . Probability density function plot before (Left) and after Box-Cox transformation. Transformed data points (Blue) are considerably closer to the fitted Normal distribution line (Green).
    Figure Legend Snippet: Primary cilia inmunolabeling and measurement. a. Confluent monolayers of wild-type LLC-PK1 cells were labeled with an anti-acetylated α-tubulin antibody (Green, Left Panel), and DAPI (Blue, Middle Panel). The merged image is shown on the Right. b. Primary cilia were identified (x40) and measured with the IPLab software. c. Top. Data distribution (Frequency) histograms before (Left panel) and after the Box-Cox transformation for the variable “Length of the primary cilium” are shown before and after Box-Cox transformation. The symmetry reached after the transformation is consistent with a Normalized distribution. Bottom . Probability density function plot before (Left) and after Box-Cox transformation. Transformed data points (Blue) are considerably closer to the fitted Normal distribution line (Green).

    Techniques Used: Labeling, Software, Transformation Assay

    Effect of PC2 channel inhibitors on the length of the primary cilium of wild type LLC-PK1 cells. a. Acetylated-α-tubulin antibody immunolabeling of LLC-PK1 cells (FITC, Green) to observe the length of primary cilia under control (Ctrl, Top Panel) condition, and the presence of either amiloride (200 μM, Middle Panel), or LiCl (10 mM, Bottom Panel). Cells were incubated in normal 1.2 mM Ca 2+ . Images were obtained with x60 objective. b. Histograms of ciliary length measurements in normal Ca 2+ and the presence of either amiloride (Top) or LiCl (Bottom) are shown before (Left), and after (Right), Box-Cox transformation. A not Normal left-skewed distribution of data is observed before transformation. c. Bar graphs of percentage increase (positive bars) or decrease (negative bars) in the length of the primary cilium under various conditions, including Free- and high (6.2 mM) Ca 2+ external condition, as well as incubations with LiCl (10 mM) and amiloride (200 μM) respect to 1.2 Ca condition. Li + and amiloride were added in the presence of external 1.2 mM Ca 2+ . All data are the mean ± SEM of percentage change as compared with respect to the control condition in normal Ca 2+ . Asterisks indicate statistical significance: *** p
    Figure Legend Snippet: Effect of PC2 channel inhibitors on the length of the primary cilium of wild type LLC-PK1 cells. a. Acetylated-α-tubulin antibody immunolabeling of LLC-PK1 cells (FITC, Green) to observe the length of primary cilia under control (Ctrl, Top Panel) condition, and the presence of either amiloride (200 μM, Middle Panel), or LiCl (10 mM, Bottom Panel). Cells were incubated in normal 1.2 mM Ca 2+ . Images were obtained with x60 objective. b. Histograms of ciliary length measurements in normal Ca 2+ and the presence of either amiloride (Top) or LiCl (Bottom) are shown before (Left), and after (Right), Box-Cox transformation. A not Normal left-skewed distribution of data is observed before transformation. c. Bar graphs of percentage increase (positive bars) or decrease (negative bars) in the length of the primary cilium under various conditions, including Free- and high (6.2 mM) Ca 2+ external condition, as well as incubations with LiCl (10 mM) and amiloride (200 μM) respect to 1.2 Ca condition. Li + and amiloride were added in the presence of external 1.2 mM Ca 2+ . All data are the mean ± SEM of percentage change as compared with respect to the control condition in normal Ca 2+ . Asterisks indicate statistical significance: *** p

    Techniques Used: Immunolabeling, Incubation, Transformation Assay

    30) Product Images from "Protective effects of methanolic extract form fruits of Lycium ruthenicum Murr on 2,2’-azobis (2-amidinopropane) dihydrochloride-induced oxidative stress in LLC-PK1 cells"

    Article Title: Protective effects of methanolic extract form fruits of Lycium ruthenicum Murr on 2,2’-azobis (2-amidinopropane) dihydrochloride-induced oxidative stress in LLC-PK1 cells

    Journal: Pharmacognosy Magazine

    doi: 10.4103/0973-1296.141790

    Effects of Lycium ruthenicum Murr fruits methanolic extracts (LFME) on reactive oxygen species in 1 mM 2, 2’-azobis (2-amidinopropane) dihydrochloride-treated LLC-PK1 cells. Data represent mean ± SD. a~e Mean values with different letters on the bars are significantly different ( P
    Figure Legend Snippet: Effects of Lycium ruthenicum Murr fruits methanolic extracts (LFME) on reactive oxygen species in 1 mM 2, 2’-azobis (2-amidinopropane) dihydrochloride-treated LLC-PK1 cells. Data represent mean ± SD. a~e Mean values with different letters on the bars are significantly different ( P

    Techniques Used:

    Effects of Lycium ruthenicum Murr fruits methanolic extracts (LFME) on cell viability in 1 mM 2, 2’-azobis(2-amidinopropane) dihydrochloride-treated LLC-PK1 cells. Data represent mean ± SD. a~f Mean values with different letters on the bars are significantly different ( P
    Figure Legend Snippet: Effects of Lycium ruthenicum Murr fruits methanolic extracts (LFME) on cell viability in 1 mM 2, 2’-azobis(2-amidinopropane) dihydrochloride-treated LLC-PK1 cells. Data represent mean ± SD. a~f Mean values with different letters on the bars are significantly different ( P

    Techniques Used:

    Effects of Lycium ruthenicum Murr fruits methanolic extracts on malondialdehyde in 1 mM 2, 2’-azobis (2-amidinopropane) dihydrochloride-treated LLC-PK1 cells. Data represent mean ± SD. a~g Mean values with different letters on the bars are significantly different ( P
    Figure Legend Snippet: Effects of Lycium ruthenicum Murr fruits methanolic extracts on malondialdehyde in 1 mM 2, 2’-azobis (2-amidinopropane) dihydrochloride-treated LLC-PK1 cells. Data represent mean ± SD. a~g Mean values with different letters on the bars are significantly different ( P

    Techniques Used:

    Effects of Lycium ruthenicum Murr fruits methanolic extracts on glutathione in 1 mM 2,2’-azobis(2-amidinopropane) dihydrochloride-treated LLC-PK1 cells. Data represent mean ± SD. a~e Mean values with different letters on the bars are significantly different ( P
    Figure Legend Snippet: Effects of Lycium ruthenicum Murr fruits methanolic extracts on glutathione in 1 mM 2,2’-azobis(2-amidinopropane) dihydrochloride-treated LLC-PK1 cells. Data represent mean ± SD. a~e Mean values with different letters on the bars are significantly different ( P

    Techniques Used:

    31) Product Images from "Isolation, genomic characterization, and pathogenicity of a Chinese porcine deltacoronavirus strain CHN-HN-2014"

    Article Title: Isolation, genomic characterization, and pathogenicity of a Chinese porcine deltacoronavirus strain CHN-HN-2014

    Journal: Veterinary Microbiology

    doi: 10.1016/j.vetmic.2016.10.022

    Cytopathic effect and IFA staining of LLC-PK1 cells infected with PDCoV strain CHN-HN-2014. (A, B and C) Cytopathic effect (CPE) in LLC-PK1 cells at 12 h (A), 24 h (B), and 36 h (C) after PDCoV CHN-HN-2014 infection. (E, F and G) Morphology of the control LLC-PK1 cells, without PDCoV infection, at 12 h (E), 24 h (F), and 36 h (G). (D, H) LLC-PK1 cells were infected (D) or mock-infected with PDCoV CHN-HN-2014. At 24 h postinfection, an immunofluorescence assay (IFA) was performed with a monoclonal antibody against PDCoV N protein. Bar, 50 μm.
    Figure Legend Snippet: Cytopathic effect and IFA staining of LLC-PK1 cells infected with PDCoV strain CHN-HN-2014. (A, B and C) Cytopathic effect (CPE) in LLC-PK1 cells at 12 h (A), 24 h (B), and 36 h (C) after PDCoV CHN-HN-2014 infection. (E, F and G) Morphology of the control LLC-PK1 cells, without PDCoV infection, at 12 h (E), 24 h (F), and 36 h (G). (D, H) LLC-PK1 cells were infected (D) or mock-infected with PDCoV CHN-HN-2014. At 24 h postinfection, an immunofluorescence assay (IFA) was performed with a monoclonal antibody against PDCoV N protein. Bar, 50 μm.

    Techniques Used: Immunofluorescence, Staining, Infection

    32) Product Images from "Twinfilin 2 regulates actin filament lengths in cochlear stereocilia"

    Article Title: Twinfilin 2 regulates actin filament lengths in cochlear stereocilia

    Journal: The Journal of neuroscience : the official journal of the Society for Neuroscience

    doi: 10.1523/JNEUROSCI.2782-09.2009

    Twinfilin 2 transfection of CL4 cells (A) CL4 cells stably expressing GFP-espin (green) were tranfected with a vector containing twinfilin 2 IRES mCherry. Red identifies mCherry expression. (B) Higher magnification of the area indicated with the white box in (A) with the dotted line pointing out the border between the transfected and untransfected cells. (C,D) Microvilli length changes of control transfected cells in comparison with neighboring untransfected cells. Microvilli lengths of control-transfected cells expressing mCherry only (red) did not change. Scale bars = 5μm.
    Figure Legend Snippet: Twinfilin 2 transfection of CL4 cells (A) CL4 cells stably expressing GFP-espin (green) were tranfected with a vector containing twinfilin 2 IRES mCherry. Red identifies mCherry expression. (B) Higher magnification of the area indicated with the white box in (A) with the dotted line pointing out the border between the transfected and untransfected cells. (C,D) Microvilli length changes of control transfected cells in comparison with neighboring untransfected cells. Microvilli lengths of control-transfected cells expressing mCherry only (red) did not change. Scale bars = 5μm.

    Techniques Used: Transfection, Stable Transfection, Expressing, Plasmid Preparation

    33) Product Images from "Chemical Characterization of a Renoprotective Metabolite from Termite-Associated Streptomyces sp. RB1 against Cisplatin-Induced Cytotoxicity"

    Article Title: Chemical Characterization of a Renoprotective Metabolite from Termite-Associated Streptomyces sp. RB1 against Cisplatin-Induced Cytotoxicity

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms19010174

    Protective effect of ABR on apoptosis in LLC-PK1 cells exposed to 25 μM cisplatin for 24 h by western blot ( A ) Expression levels of MAPK-caspase-3 pathway proteins; ( B ) Each bar graph represents densitometric quantification of western blot bands. Control cells were treated with the vehicle only (mean ± SD, * p
    Figure Legend Snippet: Protective effect of ABR on apoptosis in LLC-PK1 cells exposed to 25 μM cisplatin for 24 h by western blot ( A ) Expression levels of MAPK-caspase-3 pathway proteins; ( B ) Each bar graph represents densitometric quantification of western blot bands. Control cells were treated with the vehicle only (mean ± SD, * p

    Techniques Used: Western Blot, Expressing

    Protective effect of ABR on apoptosis in LLC-PK1 cells exposed to 25 μM cisplatin for 24 h by image-based cytometric assay. ( A ) Representative images for apoptosis detection (green) (Magnification: 4×); ( B ) Percentage of Annexin V-positive-stained apoptotic cells. Control cells were treated with the vehicle only (mean ± SD, * p
    Figure Legend Snippet: Protective effect of ABR on apoptosis in LLC-PK1 cells exposed to 25 μM cisplatin for 24 h by image-based cytometric assay. ( A ) Representative images for apoptosis detection (green) (Magnification: 4×); ( B ) Percentage of Annexin V-positive-stained apoptotic cells. Control cells were treated with the vehicle only (mean ± SD, * p

    Techniques Used: Staining

    Protective effects of the MeOH extract (ME) of Streptomyces sp. RB1 and its fractions (hexane-soluble (H), CH 2 Cl 2 -soluble (C), EtOAc-soluble (EA), and n -BuOH-soluble fractions (BU)) in LLC-PK1 cells exposed to 25 μM of cisplatin for 24 h by MTT assay. Protective effects of ( A ) ME, and ( B ) H fraction, ( C ) C fraction, ( D ) EA fraction, and ( E ) BU fraction in LLC-PK1 cells exposed to 25 μM of cisplatin for 24 h by MTT assay. The concentrations of samples were 0, 10, 50, 100 and 250 μg/mL. Control cells were treated with the vehicle only (mean ± SD, * p
    Figure Legend Snippet: Protective effects of the MeOH extract (ME) of Streptomyces sp. RB1 and its fractions (hexane-soluble (H), CH 2 Cl 2 -soluble (C), EtOAc-soluble (EA), and n -BuOH-soluble fractions (BU)) in LLC-PK1 cells exposed to 25 μM of cisplatin for 24 h by MTT assay. Protective effects of ( A ) ME, and ( B ) H fraction, ( C ) C fraction, ( D ) EA fraction, and ( E ) BU fraction in LLC-PK1 cells exposed to 25 μM of cisplatin for 24 h by MTT assay. The concentrations of samples were 0, 10, 50, 100 and 250 μg/mL. Control cells were treated with the vehicle only (mean ± SD, * p

    Techniques Used: MTT Assay

    34) Product Images from "Inhibition of Intracellular ROS Accumulation by Formononetin Attenuates Cisplatin-Mediated Apoptosis in LLC-PK1 Cells"

    Article Title: Inhibition of Intracellular ROS Accumulation by Formononetin Attenuates Cisplatin-Mediated Apoptosis in LLC-PK1 Cells

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms19030813

    Formononetin attenuates cisplatin-induced apoptosis in LLC-PK1 cells. ( a ) LLC-PK1 cells were exposed to 25 μM cisplatin for 24 h in the presence of formononetin followed by staining with Hoechst-33342. Bright blue indicates the chromatin condensation. Scale bar, 50 μm; ( b ) representative images (40× magnification) were obtained using Tali-image–based cytometric analysis to determine the apoptotic cells (blue: live cells, green color: Annexin V positive cells); ( c ) bars indicate the percentage of apoptotic cells (mean ± S.E.M., * p
    Figure Legend Snippet: Formononetin attenuates cisplatin-induced apoptosis in LLC-PK1 cells. ( a ) LLC-PK1 cells were exposed to 25 μM cisplatin for 24 h in the presence of formononetin followed by staining with Hoechst-33342. Bright blue indicates the chromatin condensation. Scale bar, 50 μm; ( b ) representative images (40× magnification) were obtained using Tali-image–based cytometric analysis to determine the apoptotic cells (blue: live cells, green color: Annexin V positive cells); ( c ) bars indicate the percentage of apoptotic cells (mean ± S.E.M., * p

    Techniques Used: Staining

    Formononetin prevents cisplatin-induced LLC-PK1 cell death. ( a ) LLC-PK1 cells were treated with the indicated concentrations of formononetin and cell viability was determined. Bars denote the percentage of cell viability (mean ± S.E.M.). ( b ) LLC-PK1 cells were treated with the indicated concentrations of cisplatin (Cis) and cell viability was determined. Bars denote the percentage of cell viability (mean ± S.E.M., * p
    Figure Legend Snippet: Formononetin prevents cisplatin-induced LLC-PK1 cell death. ( a ) LLC-PK1 cells were treated with the indicated concentrations of formononetin and cell viability was determined. Bars denote the percentage of cell viability (mean ± S.E.M.). ( b ) LLC-PK1 cells were treated with the indicated concentrations of cisplatin (Cis) and cell viability was determined. Bars denote the percentage of cell viability (mean ± S.E.M., * p

    Techniques Used:

    Formononetin inhibits cisplatin-induced oxidative stress. ( a ) LLC-PK1 cells were treated with 25 μM cisplatin in the presence or absence of the NAC and carried out cell viability assay. Bars denotes the percentage of cell viability (mean ± S.E.M., * p
    Figure Legend Snippet: Formononetin inhibits cisplatin-induced oxidative stress. ( a ) LLC-PK1 cells were treated with 25 μM cisplatin in the presence or absence of the NAC and carried out cell viability assay. Bars denotes the percentage of cell viability (mean ± S.E.M., * p

    Techniques Used: Viability Assay

    35) Product Images from "Benzyl salicylate from the stems and stem barks of Cornus walteri as a nephroprotective agent against cisplatin-induced apoptotic cell death in LLC-PK1 cells †"

    Article Title: Benzyl salicylate from the stems and stem barks of Cornus walteri as a nephroprotective agent against cisplatin-induced apoptotic cell death in LLC-PK1 cells †

    Journal: RSC Advances

    doi: 10.1039/c9ra07009e

    Effects of BS on the levels of p-JNK, p-p38, and p-ERK in LLC-PK1 cells with cisplatin-induced damage. Immunoreactive bands of (A) p-JNK, p-p38, and p-ERK detected using western blot analyses. (B) Quantitative analyses of relative protein levels. * p
    Figure Legend Snippet: Effects of BS on the levels of p-JNK, p-p38, and p-ERK in LLC-PK1 cells with cisplatin-induced damage. Immunoreactive bands of (A) p-JNK, p-p38, and p-ERK detected using western blot analyses. (B) Quantitative analyses of relative protein levels. * p

    Techniques Used: Western Blot

    Effects of BS on the levels of Bcl-2, Bax, cleaved caspase-3, -9, and -8 in LLC PK1 cells with cisplatin-induced damage. Immunoreactive bands of (A) Bcl-2, Bax, and cleaved caspase-8, -9, and -3 detected using western blot analyses. (B) Quantitative analyses of relative protein levels. * p
    Figure Legend Snippet: Effects of BS on the levels of Bcl-2, Bax, cleaved caspase-3, -9, and -8 in LLC PK1 cells with cisplatin-induced damage. Immunoreactive bands of (A) Bcl-2, Bax, and cleaved caspase-8, -9, and -3 detected using western blot analyses. (B) Quantitative analyses of relative protein levels. * p

    Techniques Used: Western Blot

    Chemical structures of compounds 1–14 (A) and benzyl salicylate (BS) (15) (B). Protective effects of 6β-hydroxysitostenone (HS) (8) and BS (15), and quercetin (positive control, (E)) on the viability of cisplatin-damaged LLC-PK1 cells for 24 h, evaluated via the MTT assay (C and D). * p
    Figure Legend Snippet: Chemical structures of compounds 1–14 (A) and benzyl salicylate (BS) (15) (B). Protective effects of 6β-hydroxysitostenone (HS) (8) and BS (15), and quercetin (positive control, (E)) on the viability of cisplatin-damaged LLC-PK1 cells for 24 h, evaluated via the MTT assay (C and D). * p

    Techniques Used: Positive Control, MTT Assay

    Effects of benzyl salicylate (BS) on cisplatin-induced morphological changes and nuclear condensation in LLC-PK1 cells. (A) Representative phase contrast microscopy images of cells. (B) Representative fluorescence microscopy images of cells stained with Hoechst 33342. Scale bar, 50 μm.
    Figure Legend Snippet: Effects of benzyl salicylate (BS) on cisplatin-induced morphological changes and nuclear condensation in LLC-PK1 cells. (A) Representative phase contrast microscopy images of cells. (B) Representative fluorescence microscopy images of cells stained with Hoechst 33342. Scale bar, 50 μm.

    Techniques Used: Microscopy, Fluorescence, Staining

    Effect of BS on cisplatin-induced apoptosis in LLC-PK1 cells. (A) Visualization of apoptotic (green circles), live, and necrotic cells (red circles) cells (40× magnification). (B) Bar graph showing percentage of percentage of apoptotic, live, and necrotic cells. * p
    Figure Legend Snippet: Effect of BS on cisplatin-induced apoptosis in LLC-PK1 cells. (A) Visualization of apoptotic (green circles), live, and necrotic cells (red circles) cells (40× magnification). (B) Bar graph showing percentage of percentage of apoptotic, live, and necrotic cells. * p

    Techniques Used:

    Effect of BS on cisplatin-induced oxidative stress in LLC-PK1 cells. (A) Accumulation of ROS visualized with H 2 DCFDA. Scale bar, 50 μm. (B) Bar graph showing the fold increase in the intracellular ROS accumulation. * p
    Figure Legend Snippet: Effect of BS on cisplatin-induced oxidative stress in LLC-PK1 cells. (A) Accumulation of ROS visualized with H 2 DCFDA. Scale bar, 50 μm. (B) Bar graph showing the fold increase in the intracellular ROS accumulation. * p

    Techniques Used:

    36) Product Images from "Defining in vivo dose‐response curves for kidney DNA adduct formation of aristolochic acid I in rat, mouse and human by an in vitro and physiologically based kinetic modeling approach. Defining in vivo dose‐response curves for kidney DNA adduct formation of aristolochic acid I in rat, mouse and human by an in vitro and physiologically based kinetic modeling approach"

    Article Title: Defining in vivo dose‐response curves for kidney DNA adduct formation of aristolochic acid I in rat, mouse and human by an in vitro and physiologically based kinetic modeling approach. Defining in vivo dose‐response curves for kidney DNA adduct formation of aristolochic acid I in rat, mouse and human by an in vitro and physiologically based kinetic modeling approach

    Journal: Journal of Applied Toxicology

    doi: 10.1002/jat.4024

    Deoxyadenosine‐AAI adduct formation in LLC‐PK1 kidney cell line expressed in number of adducts/10 8 nt as a function of AUC AAI(in vitro) (h × μmol/L), and quantified by liquid chromatography‐electrospray ionization‐tandem mass spectrometry (mean ± SD). Linear equation was fit through the origin. AAI, aristolochic acid I; AUC, area under the curve; nts, nucleotides
    Figure Legend Snippet: Deoxyadenosine‐AAI adduct formation in LLC‐PK1 kidney cell line expressed in number of adducts/10 8 nt as a function of AUC AAI(in vitro) (h × μmol/L), and quantified by liquid chromatography‐electrospray ionization‐tandem mass spectrometry (mean ± SD). Linear equation was fit through the origin. AAI, aristolochic acid I; AUC, area under the curve; nts, nucleotides

    Techniques Used: Liquid Chromatography, Mass Spectrometry

    Concentration‐response curves for dA‐AAI (circle) and dG‐AAI adduct formation (triangle) in LLC‐PK1 cells upon 24 h exposure to increasing concentrations of AAI (μ m ). A, Expressed in number of adducts/10 8 nt as quantified by LC‐ESI‐MS/MS (mean ± SD). B, Comparison with concentration‐response curves reported in literature quantified by 32 P‐postlabeling. Black symbols show data from kidney cell lines and gray symbols show data from nonkidney cell lines. Linear equation was fit through the origin. AAI, aristolochic acid I; dA‐AAI, deoxyadenosine AAI; dG‐AAI, deoxyguanosine AAI; FCS, fetal calf serum; nts, nucleotides
    Figure Legend Snippet: Concentration‐response curves for dA‐AAI (circle) and dG‐AAI adduct formation (triangle) in LLC‐PK1 cells upon 24 h exposure to increasing concentrations of AAI (μ m ). A, Expressed in number of adducts/10 8 nt as quantified by LC‐ESI‐MS/MS (mean ± SD). B, Comparison with concentration‐response curves reported in literature quantified by 32 P‐postlabeling. Black symbols show data from kidney cell lines and gray symbols show data from nonkidney cell lines. Linear equation was fit through the origin. AAI, aristolochic acid I; dA‐AAI, deoxyadenosine AAI; dG‐AAI, deoxyguanosine AAI; FCS, fetal calf serum; nts, nucleotides

    Techniques Used: Concentration Assay, Tandem Mass Spectroscopy

    37) Product Images from "Resveratrol attenuates cerebral ischaemia reperfusion injury via modulating mitochondrial dynamics homeostasis and activating AMPK‐Mfn1 pathway. Resveratrol attenuates cerebral ischaemia reperfusion injury via modulating mitochondrial dynamics homeostasis and activating AMPK‐Mfn1 pathway"

    Article Title: Resveratrol attenuates cerebral ischaemia reperfusion injury via modulating mitochondrial dynamics homeostasis and activating AMPK‐Mfn1 pathway. Resveratrol attenuates cerebral ischaemia reperfusion injury via modulating mitochondrial dynamics homeostasis and activating AMPK‐Mfn1 pathway

    Journal: International Journal of Experimental Pathology

    doi: 10.1111/iep.12336

    Resveratrol sustains N2a cell viability in response to hypoxia‐reoxygenation (HR) injury. A, MTT assay was used to confirm the alterations of N2a cell viability. Resveratrol was added into the medium of N2a cell at low and high dose. N2a cell was treated with HR injury. B, Lactate dehydrogenase (LDH) release assay was used to detect cell death in answer to HR injury. Low dose and high dose of resveratrol were used to incubate with N2a cell in the presence of HR injury. C‐D, TUNEL assay for apoptotic cell. The apoptotic N2a cells were stained by TUNEL‐positive cells, and then, the number of TUNEL‐positive cell was recorded. E, Caspase‐3 activity was measured using ELISA. Resveratrol was added into the medium of N2a cell at low and high dose. N2a cell was treated with HR injury. * P
    Figure Legend Snippet: Resveratrol sustains N2a cell viability in response to hypoxia‐reoxygenation (HR) injury. A, MTT assay was used to confirm the alterations of N2a cell viability. Resveratrol was added into the medium of N2a cell at low and high dose. N2a cell was treated with HR injury. B, Lactate dehydrogenase (LDH) release assay was used to detect cell death in answer to HR injury. Low dose and high dose of resveratrol were used to incubate with N2a cell in the presence of HR injury. C‐D, TUNEL assay for apoptotic cell. The apoptotic N2a cells were stained by TUNEL‐positive cells, and then, the number of TUNEL‐positive cell was recorded. E, Caspase‐3 activity was measured using ELISA. Resveratrol was added into the medium of N2a cell at low and high dose. N2a cell was treated with HR injury. * P

    Techniques Used: MTT Assay, Lactate Dehydrogenase Assay, TUNEL Assay, Staining, Activity Assay, Enzyme-linked Immunosorbent Assay

    Resveratrol improves mitochondrial energy metabolism in hypoxia‐reoxygenation (HR)‐treated N2a cells. A‐B, JC‐1 probe was used to label mitochondrial membrane potential. Red fluorescence intensity indicates the normal mitochondrial membrane potential and green fluorescence means the damage mitochondrial membrane potential. C, Adenosine triphosphate (ATP) production was determined via ELISA. Resveratrol was added into the medium of N2a cell at low and high dose. N2a cell was treated with HR injury. D‐F, Mitochondrial respiratory complex activity was detected via ELISA. HR injury elevated the activity of mitochondrial respiratory complex activity and this effect could be inhibited by resveratrol treatment. G‐H, The uptake of glucose and lactic acid production was measured using ELISA. Resveratrol was added into the medium of N2a cell at low and high dose. N2a cell was treated with HR injury. * P
    Figure Legend Snippet: Resveratrol improves mitochondrial energy metabolism in hypoxia‐reoxygenation (HR)‐treated N2a cells. A‐B, JC‐1 probe was used to label mitochondrial membrane potential. Red fluorescence intensity indicates the normal mitochondrial membrane potential and green fluorescence means the damage mitochondrial membrane potential. C, Adenosine triphosphate (ATP) production was determined via ELISA. Resveratrol was added into the medium of N2a cell at low and high dose. N2a cell was treated with HR injury. D‐F, Mitochondrial respiratory complex activity was detected via ELISA. HR injury elevated the activity of mitochondrial respiratory complex activity and this effect could be inhibited by resveratrol treatment. G‐H, The uptake of glucose and lactic acid production was measured using ELISA. Resveratrol was added into the medium of N2a cell at low and high dose. N2a cell was treated with HR injury. * P

    Techniques Used: Fluorescence, Enzyme-linked Immunosorbent Assay, Activity Assay

    Mitochondrial apoptosis is inhibited by resveratrol in N2a cell. A, Caspase‐9 activity was determined using ELISA. Resveratrol was added into the medium of N2a cell at low and high dose. N2a cell was treated with hypoxia‐reoxygenation (HR) injury. B‐F, Western blotting was performed to observe the changes in mitochondrial apoptosis‐related proteins. Pro‐apoptotic proteins such as Bax and caspase‐9 were upregulated in response to HR injury and were reduced to near‐normal levels with resveratrol treatment. In contrast, anti‐apoptotic proteins such as Bcl‐2 and survivin were inhibited by HR injury and were reversed to near‐normal levels with resveratrol treatment. G‐H, Immunofluorescence assay for cyt‐c translocation from cytoplasm into nucleus. The expression of nuclear cyt‐c was determined. I, Mitochondrial permeability transition pore (mPTP) opening rate was determined using ELISA, and the mPTP opening was negatively modulated by resveratrol in the setting of cerebral IR injury. * P
    Figure Legend Snippet: Mitochondrial apoptosis is inhibited by resveratrol in N2a cell. A, Caspase‐9 activity was determined using ELISA. Resveratrol was added into the medium of N2a cell at low and high dose. N2a cell was treated with hypoxia‐reoxygenation (HR) injury. B‐F, Western blotting was performed to observe the changes in mitochondrial apoptosis‐related proteins. Pro‐apoptotic proteins such as Bax and caspase‐9 were upregulated in response to HR injury and were reduced to near‐normal levels with resveratrol treatment. In contrast, anti‐apoptotic proteins such as Bcl‐2 and survivin were inhibited by HR injury and were reversed to near‐normal levels with resveratrol treatment. G‐H, Immunofluorescence assay for cyt‐c translocation from cytoplasm into nucleus. The expression of nuclear cyt‐c was determined. I, Mitochondrial permeability transition pore (mPTP) opening rate was determined using ELISA, and the mPTP opening was negatively modulated by resveratrol in the setting of cerebral IR injury. * P

    Techniques Used: Activity Assay, Enzyme-linked Immunosorbent Assay, Western Blot, Immunofluorescence, Translocation Assay, Expressing, Permeability

    Mfn1‐mediated mitochondrial protection system is activated by resveratrol in the presence of hypoxia‐reoxygenation (HR) injury. A‐B, Western blotting was used to observe the alterations of Mfn1 expression in response to HR injury and/or resveratrol treatment. Besides, siRNA against Mfn1 was transfected into N2a cell and the knockdown efficiency of N2a cell was determined via Western blotting. C, Cell viability was measured via MTT assay. D, Lactate dehydrogenase (LDH) release assay was used to verify the cell death in response to Mfn1 knockdown. E, Adenosine triphosphate (ATP) production was detected via ELISA. Resveratrol was added into the medium of N2a cell at low and high dose. N2a cell was treated with HR injury. F, Caspase‐9 activity was measured to reflect the mitochondrial damage in response to Mfn1 deletion. * P
    Figure Legend Snippet: Mfn1‐mediated mitochondrial protection system is activated by resveratrol in the presence of hypoxia‐reoxygenation (HR) injury. A‐B, Western blotting was used to observe the alterations of Mfn1 expression in response to HR injury and/or resveratrol treatment. Besides, siRNA against Mfn1 was transfected into N2a cell and the knockdown efficiency of N2a cell was determined via Western blotting. C, Cell viability was measured via MTT assay. D, Lactate dehydrogenase (LDH) release assay was used to verify the cell death in response to Mfn1 knockdown. E, Adenosine triphosphate (ATP) production was detected via ELISA. Resveratrol was added into the medium of N2a cell at low and high dose. N2a cell was treated with HR injury. F, Caspase‐9 activity was measured to reflect the mitochondrial damage in response to Mfn1 deletion. * P

    Techniques Used: Western Blot, Expressing, Transfection, MTT Assay, Lactate Dehydrogenase Assay, Enzyme-linked Immunosorbent Assay, Activity Assay

    Resveratrol treatment attenuates hypoxia‐reoxygenation (HR)‐mediated mitochondrial oxidative stress in N2a cell. A‐B, Mitochondrial oxidative stress was evaluated via immunofluorescence. C‐E, ELISA was used to measure the change in cellular antioxidants such as superoxide dismutase (SOD), glutathione (GSH) and glutathione peroxidase (GPX). * P
    Figure Legend Snippet: Resveratrol treatment attenuates hypoxia‐reoxygenation (HR)‐mediated mitochondrial oxidative stress in N2a cell. A‐B, Mitochondrial oxidative stress was evaluated via immunofluorescence. C‐E, ELISA was used to measure the change in cellular antioxidants such as superoxide dismutase (SOD), glutathione (GSH) and glutathione peroxidase (GPX). * P

    Techniques Used: Immunofluorescence, Enzyme-linked Immunosorbent Assay

    38) Product Images from "Development and utilization of an infectious clone for porcine deltacoronavirus strain USA/IL/2014/026"

    Article Title: Development and utilization of an infectious clone for porcine deltacoronavirus strain USA/IL/2014/026

    Journal: Virology

    doi: 10.1016/j.virol.2020.11.002

    Characterization of the rescued PDCoV from the infectious clone. (A) Representative plaques of mock-, the parental strain-, and icPDCoV-infected LLC-PK1 cells at 40 h post-infection. (B) PCR fragment amplified from the parental virus was digested by the Sap I enzyme, while the PCR product derived from icPDCoV was resistant to Sap I-digestion. (C) Growth kinetics of the parental strain (Parental) and infectious clone strain (icPDCoV) in PK1 cells. (D) Detection of N protein expression in parental- or icPDCoV-infected PK1 cells using an immunofluorescence assay with a mouse anti -N monoclonal antibody.
    Figure Legend Snippet: Characterization of the rescued PDCoV from the infectious clone. (A) Representative plaques of mock-, the parental strain-, and icPDCoV-infected LLC-PK1 cells at 40 h post-infection. (B) PCR fragment amplified from the parental virus was digested by the Sap I enzyme, while the PCR product derived from icPDCoV was resistant to Sap I-digestion. (C) Growth kinetics of the parental strain (Parental) and infectious clone strain (icPDCoV) in PK1 cells. (D) Detection of N protein expression in parental- or icPDCoV-infected PK1 cells using an immunofluorescence assay with a mouse anti -N monoclonal antibody.

    Techniques Used: Infection, Polymerase Chain Reaction, Amplification, Derivative Assay, Expressing, Immunofluorescence

    39) Product Images from "A multi-omics study to investigate tacrolimus nephrotoxicity mechanisms"

    Article Title: A multi-omics study to investigate tacrolimus nephrotoxicity mechanisms

    Journal: bioRxiv

    doi: 10.1101/2021.07.29.454229

    LLC-PK1 viability after incubation with 0.5% ethanol (control (C)) or 5µM of tacrolimus (TAC) for 24h, 48h and 72h, assessed using the MTS viability assay (n=18, left graph) and annexin 5/ 7AAD staining (n=5, right graph). Graphs represent the % of cell viability with tacrolimus as compared to control for each incubation duration. Statistical analysis with the t-test. *** p
    Figure Legend Snippet: LLC-PK1 viability after incubation with 0.5% ethanol (control (C)) or 5µM of tacrolimus (TAC) for 24h, 48h and 72h, assessed using the MTS viability assay (n=18, left graph) and annexin 5/ 7AAD staining (n=5, right graph). Graphs represent the % of cell viability with tacrolimus as compared to control for each incubation duration. Statistical analysis with the t-test. *** p

    Techniques Used: Incubation, Viability Assay, Staining

    40) Product Images from "Correlative microscopy of freeze-dried cells and studies on intracellular calcium stores with imaging secondary ion mass spectrometry (SIMS)"

    Article Title: Correlative microscopy of freeze-dried cells and studies on intracellular calcium stores with imaging secondary ion mass spectrometry (SIMS)

    Journal: Journal of analytical atomic spectrometry

    doi: 10.1039/c9ja00193j

    SIMS images of 44 Ca influx and endogenous 40 Ca calcium in resting and stimulated renal epithelial LLC-PK1 cells. The cells were exposed for 5 minutes to a buffer containing 1.8 mM of the stable isotope 44 Ca in the presence of 1 µM of the calcium ionophore A23187 (middle row) and 100 nM of the arginine vasopressin hormone (bottom row).
    Figure Legend Snippet: SIMS images of 44 Ca influx and endogenous 40 Ca calcium in resting and stimulated renal epithelial LLC-PK1 cells. The cells were exposed for 5 minutes to a buffer containing 1.8 mM of the stable isotope 44 Ca in the presence of 1 µM of the calcium ionophore A23187 (middle row) and 100 nM of the arginine vasopressin hormone (bottom row).

    Techniques Used:

    SIMS images of 39 K, 23 Na, and 40 Ca revealing intracellular distributions of potassium, sodium, and calcium in renal epithelial LLC-PK1 cells. The cells were treated with 1 µM nocodazole for 4 hours and 100 nM taxol for 18 hours.
    Figure Legend Snippet: SIMS images of 39 K, 23 Na, and 40 Ca revealing intracellular distributions of potassium, sodium, and calcium in renal epithelial LLC-PK1 cells. The cells were treated with 1 µM nocodazole for 4 hours and 100 nM taxol for 18 hours.

    Techniques Used:

    SIMS images showing the distributions of 39 K, 23 Na, 40 Ca, and 12 C in fractured freeze-dried renal epithelial LLC-PK1 cells.
    Figure Legend Snippet: SIMS images showing the distributions of 39 K, 23 Na, 40 Ca, and 12 C in fractured freeze-dried renal epithelial LLC-PK1 cells.

    Techniques Used:

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    ATCC llc pk1
    Role of p38 in inhibiting PDCoV replication and alleviating PDCoV-induced apoptosis by EP in <t>LLC-PK1</t> cells. A The efficiency of p38 siRNA was evaluated by Western blot. LLC-PK1 cells were transfected with the indicated siRNA. At 24 h post-transfection, the expression of p38 was analyzed by Western blot. Results were presented as the ratio of p38 band intensity to β-actin band intensity. B LLC-PK1 cells grown in 6-well plates were transfected with p38 siRNA #1 or NC siRNA for 24 h, and then infected with 2 MOI PDCoV or mock-infected in the absence or presence EP (150 μM). After PDCoV adsorption for 1 h, the cells were further cultured in fresh medium in the absence or presence EP (150 μM). Western blot analysis of proteins from indicated LLC-PK1 cells probed with the anti-p-p38, anti-p38, anti-cleaved caspase-3, anti-caspase-3, anti-Bax, anti-Bcl-2 and anti-PDCoV N antibody. C – I Results are presented as the ratio of target protein band intensity to β-actin band intensity. Data are presented as the mean ± SEM. * P
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    Role of p38 in inhibiting PDCoV replication and alleviating PDCoV-induced apoptosis by EP in LLC-PK1 cells. A The efficiency of p38 siRNA was evaluated by Western blot. LLC-PK1 cells were transfected with the indicated siRNA. At 24 h post-transfection, the expression of p38 was analyzed by Western blot. Results were presented as the ratio of p38 band intensity to β-actin band intensity. B LLC-PK1 cells grown in 6-well plates were transfected with p38 siRNA #1 or NC siRNA for 24 h, and then infected with 2 MOI PDCoV or mock-infected in the absence or presence EP (150 μM). After PDCoV adsorption for 1 h, the cells were further cultured in fresh medium in the absence or presence EP (150 μM). Western blot analysis of proteins from indicated LLC-PK1 cells probed with the anti-p-p38, anti-p38, anti-cleaved caspase-3, anti-caspase-3, anti-Bax, anti-Bcl-2 and anti-PDCoV N antibody. C – I Results are presented as the ratio of target protein band intensity to β-actin band intensity. Data are presented as the mean ± SEM. * P

    Journal: Veterinary Research

    Article Title: Antiviral effects of ergosterol peroxide in a pig model of porcine deltacoronavirus (PDCoV) infection involves modulation of apoptosis and tight junction in the small intestine

    doi: 10.1186/s13567-021-00955-5

    Figure Lengend Snippet: Role of p38 in inhibiting PDCoV replication and alleviating PDCoV-induced apoptosis by EP in LLC-PK1 cells. A The efficiency of p38 siRNA was evaluated by Western blot. LLC-PK1 cells were transfected with the indicated siRNA. At 24 h post-transfection, the expression of p38 was analyzed by Western blot. Results were presented as the ratio of p38 band intensity to β-actin band intensity. B LLC-PK1 cells grown in 6-well plates were transfected with p38 siRNA #1 or NC siRNA for 24 h, and then infected with 2 MOI PDCoV or mock-infected in the absence or presence EP (150 μM). After PDCoV adsorption for 1 h, the cells were further cultured in fresh medium in the absence or presence EP (150 μM). Western blot analysis of proteins from indicated LLC-PK1 cells probed with the anti-p-p38, anti-p38, anti-cleaved caspase-3, anti-caspase-3, anti-Bax, anti-Bcl-2 and anti-PDCoV N antibody. C – I Results are presented as the ratio of target protein band intensity to β-actin band intensity. Data are presented as the mean ± SEM. * P

    Article Snippet: Cell culture LLC-PK1 cells (ATCC CL-101) were obtained from the American Type Culture Collection (ATCC) and cultured in MEM (Gibco, USA) supplemented with 1% antibiotic–antimycotic (Gibco, USA), 1% HEPES (Gibco, USA), 1% MEM non-essential amino acids solution (NEAA) (Gibco, USA) and 10% heat-inactivated fetal bovine serum (FBS) (Gibco, Australia) at 37 °C with 5% CO2.

    Techniques: Western Blot, Transfection, Expressing, Infection, Adsorption, Cell Culture

    PDCoV nsp15 antagonizes SEV-induced IFN-β production. ( A , C ) LLC-PK1 cells (A) or HEK-293T cells (C) cultured in 24-well plates were co-transfected with IFN-β-Luc and pRL-TK together with increasing amounts (0.1, 0.5, and 1.0 μg) of pCAGGS-HA-nsp15 or empty vector. After 24 h, the cells were mock-infected or infected with SEV (10 hemagglutinating activity units/well) for 12 h and subjected to a dual-luciferase reporter assay. The relative firefly luciferase activity was normalized to the Renilla luciferase activity with the untreated empty vector control value set to 1. ( B , D ) LLC-PK1 cells (B) or HEK-293T cells (D) cultured in 24-well plates were co-transfected with 1.0 μg of pCAGGS-HA-nsp15 or empty vector for 24 h, and then left untreated or infected with SEV (10 hemagglutinating activity units/well). At 8 h after infection, the cells were collected, and total RNA was extracted to detect the expression levels of IFN-β and GAPDH by SYBR Green PCR assay. ( E , F ) LLC-PK1 (E) or HEK-293T (F) cells were transfected with 1.0 μg of pCAGGS-HA-nsp15 or empty vector. At 24 h after transfection, both cell lines were infected with SEV for 12 h and the cell supernatants were collected. The UV-irradiated cell supernatants were overlaid onto fresh LLC-PK1 or HEK-293T cells in 24-well plates. After 24 h of incubation, cells were infected with VSV-GFP for 12 h. The replication of VSV-GFP was detected via fluorescence microscopy. Data are representative of three independent experiments. ***, p

    Journal: Molecular Immunology

    Article Title: Porcine deltacoronavirus nsp15 antagonizes interferon-β production independently of its endoribonuclease activity

    doi: 10.1016/j.molimm.2019.07.003

    Figure Lengend Snippet: PDCoV nsp15 antagonizes SEV-induced IFN-β production. ( A , C ) LLC-PK1 cells (A) or HEK-293T cells (C) cultured in 24-well plates were co-transfected with IFN-β-Luc and pRL-TK together with increasing amounts (0.1, 0.5, and 1.0 μg) of pCAGGS-HA-nsp15 or empty vector. After 24 h, the cells were mock-infected or infected with SEV (10 hemagglutinating activity units/well) for 12 h and subjected to a dual-luciferase reporter assay. The relative firefly luciferase activity was normalized to the Renilla luciferase activity with the untreated empty vector control value set to 1. ( B , D ) LLC-PK1 cells (B) or HEK-293T cells (D) cultured in 24-well plates were co-transfected with 1.0 μg of pCAGGS-HA-nsp15 or empty vector for 24 h, and then left untreated or infected with SEV (10 hemagglutinating activity units/well). At 8 h after infection, the cells were collected, and total RNA was extracted to detect the expression levels of IFN-β and GAPDH by SYBR Green PCR assay. ( E , F ) LLC-PK1 (E) or HEK-293T (F) cells were transfected with 1.0 μg of pCAGGS-HA-nsp15 or empty vector. At 24 h after transfection, both cell lines were infected with SEV for 12 h and the cell supernatants were collected. The UV-irradiated cell supernatants were overlaid onto fresh LLC-PK1 or HEK-293T cells in 24-well plates. After 24 h of incubation, cells were infected with VSV-GFP for 12 h. The replication of VSV-GFP was detected via fluorescence microscopy. Data are representative of three independent experiments. ***, p

    Article Snippet: 2.1 Cells, viruses, and reagents LLC-PK1 cells (porcine kidney cells) were purchased from the ATCC (ATCC CL-101) and cultured in Dulbecco's modified Eagle's medium (Invitrogen, USA) supplemented with 10% fetal bovine serum at 37 °C in a humidified 5% CO2 incubator.

    Techniques: Cell Culture, Transfection, Plasmid Preparation, Infection, Activity Assay, Luciferase, Reporter Assay, Expressing, SYBR Green Assay, Polymerase Chain Reaction, Irradiation, Incubation, Fluorescence, Microscopy

    PDCoV nsp15 impairs SEV-induced activation of NF-κB. ( A – D ) LLC-PK1 cells (A, C) or HEK-293T cells (B, D) grown in 24-well plates were co-transfected with NF-κB-Luc (A, B) or IRF3-Luc (C, D) and pRL-TK together with increasing quantities (0.1, 0.5, and 1.0 μg) of PDCoV nsp15 expression plasmid for 24 h, followed by infection with SEV or mock-infection for 12 h before luciferase reporter assays were performed. The averages of data from three independent experiments are shown. ***, p

    Journal: Molecular Immunology

    Article Title: Porcine deltacoronavirus nsp15 antagonizes interferon-β production independently of its endoribonuclease activity

    doi: 10.1016/j.molimm.2019.07.003

    Figure Lengend Snippet: PDCoV nsp15 impairs SEV-induced activation of NF-κB. ( A – D ) LLC-PK1 cells (A, C) or HEK-293T cells (B, D) grown in 24-well plates were co-transfected with NF-κB-Luc (A, B) or IRF3-Luc (C, D) and pRL-TK together with increasing quantities (0.1, 0.5, and 1.0 μg) of PDCoV nsp15 expression plasmid for 24 h, followed by infection with SEV or mock-infection for 12 h before luciferase reporter assays were performed. The averages of data from three independent experiments are shown. ***, p

    Article Snippet: 2.1 Cells, viruses, and reagents LLC-PK1 cells (porcine kidney cells) were purchased from the ATCC (ATCC CL-101) and cultured in Dulbecco's modified Eagle's medium (Invitrogen, USA) supplemented with 10% fetal bovine serum at 37 °C in a humidified 5% CO2 incubator.

    Techniques: Activation Assay, Transfection, Expressing, Plasmid Preparation, Infection, Luciferase

    PDCoV nsp15 inhibits IFN-β production independently of its endoribonuclease activity. LLC-PK1 cells ( A ) or HEK-293T cells ( B ) cultured in 24-well plates were transfected with 1.0 μg of expression plasmid (PDCoV nsp15, H219A, H234A, or K269A) or empty vector, along with IFN-β-Luc and pRL-TK for 24 h. The cells were then mock-infected or infected with SEV for 12 h and subjected to dual-luciferase reporter assays. Data are means ± SD from three independent experiments. ***, p

    Journal: Molecular Immunology

    Article Title: Porcine deltacoronavirus nsp15 antagonizes interferon-β production independently of its endoribonuclease activity

    doi: 10.1016/j.molimm.2019.07.003

    Figure Lengend Snippet: PDCoV nsp15 inhibits IFN-β production independently of its endoribonuclease activity. LLC-PK1 cells ( A ) or HEK-293T cells ( B ) cultured in 24-well plates were transfected with 1.0 μg of expression plasmid (PDCoV nsp15, H219A, H234A, or K269A) or empty vector, along with IFN-β-Luc and pRL-TK for 24 h. The cells were then mock-infected or infected with SEV for 12 h and subjected to dual-luciferase reporter assays. Data are means ± SD from three independent experiments. ***, p

    Article Snippet: 2.1 Cells, viruses, and reagents LLC-PK1 cells (porcine kidney cells) were purchased from the ATCC (ATCC CL-101) and cultured in Dulbecco's modified Eagle's medium (Invitrogen, USA) supplemented with 10% fetal bovine serum at 37 °C in a humidified 5% CO2 incubator.

    Techniques: Activity Assay, Cell Culture, Transfection, Expressing, Plasmid Preparation, Infection, Luciferase

    PDCoV nsp15 mutants lacking endoribonuclease activity also significantly inhibit SEV-induced activation of NF-κB. ( A , B ) LLC-PK1 cells (A) or HEK-293T cells (B) grown in 24-well plates were co-transfected with NF-κB–Luc together with pRL-TK and 1.0 μg of expression plasmid (PDCoV nsp15, H219A, H234A, K269A) or empty vector for 24 h, followed by stimulation with SEV for 12 h before luciferase reporter assays were performed. ***, p

    Journal: Molecular Immunology

    Article Title: Porcine deltacoronavirus nsp15 antagonizes interferon-β production independently of its endoribonuclease activity

    doi: 10.1016/j.molimm.2019.07.003

    Figure Lengend Snippet: PDCoV nsp15 mutants lacking endoribonuclease activity also significantly inhibit SEV-induced activation of NF-κB. ( A , B ) LLC-PK1 cells (A) or HEK-293T cells (B) grown in 24-well plates were co-transfected with NF-κB–Luc together with pRL-TK and 1.0 μg of expression plasmid (PDCoV nsp15, H219A, H234A, K269A) or empty vector for 24 h, followed by stimulation with SEV for 12 h before luciferase reporter assays were performed. ***, p

    Article Snippet: 2.1 Cells, viruses, and reagents LLC-PK1 cells (porcine kidney cells) were purchased from the ATCC (ATCC CL-101) and cultured in Dulbecco's modified Eagle's medium (Invitrogen, USA) supplemented with 10% fetal bovine serum at 37 °C in a humidified 5% CO2 incubator.

    Techniques: Activity Assay, Activation Assay, Transfection, Expressing, Plasmid Preparation, Luciferase

    Generation and characterization of PDCoV mutant viruses. (A) Two mutant PDCoVs were generated in this study. icEnUmut: Histidine-219 of the endoribonuclease (EndoU) of PDCoV was mutated to Alanine (H219A). icDelNS6/nG: the coding sequence of ns6 was replaced with neonGreen (nGreen) sequence. The TRS of ns6 and N gene are shown and boxed. A 20 nt coding sequence of ns6 at the 3′ end was retained to maintain the potential secondary structure of the TRS of N gene. (B) Representative plaques of the parental strain-, icPDCoV-, icEnUmut-, and icDelNS6/nG-infected PK1 cells at 32 h post-infection. (C) Growth kinetics of icPDCoV and mutant viruses. PK1 cells were infected at a dose of 0.1 TCID 50 per cell. Cell culture supernatant was collected at the indicated time points and subject to a TCID 50 assay in PK1 cells. Error bars represent mean ± standard deviation. (D) icDelNS6/nG infection in PK1 cells caused CPE (Bright) and yielded green signal (nGreen) under a fluorescence microscope. (E) Western blotting detection of the expression of N protein using mouse anti -N monoclonal antibody (mAb). β-actin served as a loading control. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

    Journal: Virology

    Article Title: Development and utilization of an infectious clone for porcine deltacoronavirus strain USA/IL/2014/026

    doi: 10.1016/j.virol.2020.11.002

    Figure Lengend Snippet: Generation and characterization of PDCoV mutant viruses. (A) Two mutant PDCoVs were generated in this study. icEnUmut: Histidine-219 of the endoribonuclease (EndoU) of PDCoV was mutated to Alanine (H219A). icDelNS6/nG: the coding sequence of ns6 was replaced with neonGreen (nGreen) sequence. The TRS of ns6 and N gene are shown and boxed. A 20 nt coding sequence of ns6 at the 3′ end was retained to maintain the potential secondary structure of the TRS of N gene. (B) Representative plaques of the parental strain-, icPDCoV-, icEnUmut-, and icDelNS6/nG-infected PK1 cells at 32 h post-infection. (C) Growth kinetics of icPDCoV and mutant viruses. PK1 cells were infected at a dose of 0.1 TCID 50 per cell. Cell culture supernatant was collected at the indicated time points and subject to a TCID 50 assay in PK1 cells. Error bars represent mean ± standard deviation. (D) icDelNS6/nG infection in PK1 cells caused CPE (Bright) and yielded green signal (nGreen) under a fluorescence microscope. (E) Western blotting detection of the expression of N protein using mouse anti -N monoclonal antibody (mAb). β-actin served as a loading control. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

    Article Snippet: Porcine kidney epithelial cells, LLC-PK1 (ATCC, CL-101), termed PK1 cells, were purchased from ATCC and grown in growth medium containing modified Eagle medium (MEM) (Corning, 10010-CV) supplemented with heat-inactivated 5% fetal calf serum (FCS) (Atlanta Biological) and 1% penicillin/streptomycin (pen/strep; HyClone).

    Techniques: Mutagenesis, Generated, Sequencing, Infection, Cell Culture, Standard Deviation, Fluorescence, Microscopy, Western Blot, Expressing

    Characterization of host immune responses in icPDCoV- and mutant viruses-infected PK1 cells. Cells were infected at a dose of 0.1 TCID 50 per cell and were harvested for total RNA extraction at indicated time points. The relative mRNA level of (A) IFN-β, N gene, (B) ISGs (OAS1, ISG15, ISG54), and TNF-α were measured using RT-qPCR. The experiment was performed three times, and the representative data are shown. Error bars represent mean ± SD and the relative fold-changes were shown above the error bars.

    Journal: Virology

    Article Title: Development and utilization of an infectious clone for porcine deltacoronavirus strain USA/IL/2014/026

    doi: 10.1016/j.virol.2020.11.002

    Figure Lengend Snippet: Characterization of host immune responses in icPDCoV- and mutant viruses-infected PK1 cells. Cells were infected at a dose of 0.1 TCID 50 per cell and were harvested for total RNA extraction at indicated time points. The relative mRNA level of (A) IFN-β, N gene, (B) ISGs (OAS1, ISG15, ISG54), and TNF-α were measured using RT-qPCR. The experiment was performed three times, and the representative data are shown. Error bars represent mean ± SD and the relative fold-changes were shown above the error bars.

    Article Snippet: Porcine kidney epithelial cells, LLC-PK1 (ATCC, CL-101), termed PK1 cells, were purchased from ATCC and grown in growth medium containing modified Eagle medium (MEM) (Corning, 10010-CV) supplemented with heat-inactivated 5% fetal calf serum (FCS) (Atlanta Biological) and 1% penicillin/streptomycin (pen/strep; HyClone).

    Techniques: Mutagenesis, Infection, RNA Extraction, Quantitative RT-PCR

    Adaptive mutations allow rescue of infectious PDCoV that produces observable cytopathic effect in PK1 cells. In order to obtain cell culture adaptive mutation(s), the parental strain (P1) was passaged for 30 times (P30) in PK1 cells. (A) Growth kinetics analysis of P1 and P30 indicates P30 grew more rapidly and reached peak titer earlier than P1. (B) Full-genome sequencing revealed six nonsynonymous nucleotide substitutions in the genome of the P30 virus. (C) Four of the six substitutions reside in the spike sequence and are indicated in the protein structure of PDCoV spike (PDB#: 6b7n). (D) Incorporating all four (icP30) or one (H236Y) of the spike substitutions (icPDCoV) allows the recovered infectious clone virus to produce CPE in PK1 cells.

    Journal: Virology

    Article Title: Development and utilization of an infectious clone for porcine deltacoronavirus strain USA/IL/2014/026

    doi: 10.1016/j.virol.2020.11.002

    Figure Lengend Snippet: Adaptive mutations allow rescue of infectious PDCoV that produces observable cytopathic effect in PK1 cells. In order to obtain cell culture adaptive mutation(s), the parental strain (P1) was passaged for 30 times (P30) in PK1 cells. (A) Growth kinetics analysis of P1 and P30 indicates P30 grew more rapidly and reached peak titer earlier than P1. (B) Full-genome sequencing revealed six nonsynonymous nucleotide substitutions in the genome of the P30 virus. (C) Four of the six substitutions reside in the spike sequence and are indicated in the protein structure of PDCoV spike (PDB#: 6b7n). (D) Incorporating all four (icP30) or one (H236Y) of the spike substitutions (icPDCoV) allows the recovered infectious clone virus to produce CPE in PK1 cells.

    Article Snippet: Porcine kidney epithelial cells, LLC-PK1 (ATCC, CL-101), termed PK1 cells, were purchased from ATCC and grown in growth medium containing modified Eagle medium (MEM) (Corning, 10010-CV) supplemented with heat-inactivated 5% fetal calf serum (FCS) (Atlanta Biological) and 1% penicillin/streptomycin (pen/strep; HyClone).

    Techniques: Cell Culture, Mutagenesis, Sequencing

    Characterization of the rescued PDCoV from the infectious clone. (A) Representative plaques of mock-, the parental strain-, and icPDCoV-infected LLC-PK1 cells at 40 h post-infection. (B) PCR fragment amplified from the parental virus was digested by the Sap I enzyme, while the PCR product derived from icPDCoV was resistant to Sap I-digestion. (C) Growth kinetics of the parental strain (Parental) and infectious clone strain (icPDCoV) in PK1 cells. (D) Detection of N protein expression in parental- or icPDCoV-infected PK1 cells using an immunofluorescence assay with a mouse anti -N monoclonal antibody.

    Journal: Virology

    Article Title: Development and utilization of an infectious clone for porcine deltacoronavirus strain USA/IL/2014/026

    doi: 10.1016/j.virol.2020.11.002

    Figure Lengend Snippet: Characterization of the rescued PDCoV from the infectious clone. (A) Representative plaques of mock-, the parental strain-, and icPDCoV-infected LLC-PK1 cells at 40 h post-infection. (B) PCR fragment amplified from the parental virus was digested by the Sap I enzyme, while the PCR product derived from icPDCoV was resistant to Sap I-digestion. (C) Growth kinetics of the parental strain (Parental) and infectious clone strain (icPDCoV) in PK1 cells. (D) Detection of N protein expression in parental- or icPDCoV-infected PK1 cells using an immunofluorescence assay with a mouse anti -N monoclonal antibody.

    Article Snippet: Porcine kidney epithelial cells, LLC-PK1 (ATCC, CL-101), termed PK1 cells, were purchased from ATCC and grown in growth medium containing modified Eagle medium (MEM) (Corning, 10010-CV) supplemented with heat-inactivated 5% fetal calf serum (FCS) (Atlanta Biological) and 1% penicillin/streptomycin (pen/strep; HyClone).

    Techniques: Infection, Polymerase Chain Reaction, Amplification, Derivative Assay, Expressing, Immunofluorescence

    LLC-PK1 viability after incubation with 0.5% ethanol (control (C)) or 5µM of tacrolimus (TAC) for 24h, 48h and 72h, assessed using the MTS viability assay (n=18, left graph) and annexin 5/ 7AAD staining (n=5, right graph). Graphs represent the % of cell viability with tacrolimus as compared to control for each incubation duration. Statistical analysis with the t-test. *** p

    Journal: bioRxiv

    Article Title: A multi-omics study to investigate tacrolimus nephrotoxicity mechanisms

    doi: 10.1101/2021.07.29.454229

    Figure Lengend Snippet: LLC-PK1 viability after incubation with 0.5% ethanol (control (C)) or 5µM of tacrolimus (TAC) for 24h, 48h and 72h, assessed using the MTS viability assay (n=18, left graph) and annexin 5/ 7AAD staining (n=5, right graph). Graphs represent the % of cell viability with tacrolimus as compared to control for each incubation duration. Statistical analysis with the t-test. *** p

    Article Snippet: LLC-PK1 were seeded in 6-well plates and expanded up to sub-confluence in the routine cell culture medium.

    Techniques: Incubation, Viability Assay, Staining